CN111542544A - Immunostimulatory antibodies for the treatment of cancer - Google Patents

Abstract

The invention provides methods of treating cancer using agonistic antibodies that specifically bind to immunostimulatory receptors, wherein the antibodies are administered in an amount and/or frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%, e.g., from about 20% to about 80%. Also provided are methods for determining human doses of such agonistic antibodies, and methods for monitoring receptor occupancy of agonistic antibodies in order to maintain effective antibody levels in, for example, a human patient. Methods of measuring soluble OX40 in a subject are also provided. Also provided are methods of treating cancer comprising administering to a subject an effective amount of an anti-OX 40 antibody and an anti-PD-1 antibody.

Description

Immunostimulatory antibodies for the treatment of cancer

Cross reference to related applications

This PCT application claims the priority benefits of U.S. provisional application nos. 62/580,346 (filed on 1/11/2017), 62/581,441 (filed on 3/11/2017), 62/581,905 (filed on 6/11/2017), 62/583,808 (filed on 9/11/2017), 62/628,207 (filed on 8/2/2018) and 62/657,616 (filed on 13/4/2018), each of which is incorporated herein by reference in its entirety.

Reference to an electronically submitted sequence Listing

The contents of the electronic sequence listing (title: 3338.115PC06_ st25. txt; size: 30.589 bytes; creation date: 2018, 10 months and 30 days) filed with this application in the form of an ASCII text file are incorporated herein in their entirety by reference.

Background

Although the targeting of many immunosuppressive receptors has been successful in cancer therapy, predicting whether antibodies directed against a particular immune receptor are effective has been made difficult by the complex interaction between stimulatory and inhibitory receptors expressed on immune cells, such as regulatory T cells, effector cells (e.g., T cells), and antigen presenting cells. In contrast to recent success with antagonistic antibodies targeting immunosuppressive receptors (e.g., nivolumab, ipilimumab), agonistic antibodies targeting immunostimulatory receptors have had little clinical success due to, for example, lack of therapeutic efficacy and/or toxicity. These antibodies are a potential source that has not yet been developed, and if one can address the reasons for their lack of efficacy/toxicity and optimize their use, it is possible to greatly expand the pool of therapeutic modalities available for combating the oncological indications. In view of the ongoing need for better therapeutic strategies for diseases such as cancer, for example by boosting immune responses such as T cell responses, optimization of the methods for activating immunostimulatory receptors would provide therapeutic benefits.

Summary of the invention

In one aspect, the application provides a method of treating cancer comprising administering to a subject in need thereof an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the antibody is administered in an amount or frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

In another aspect, the application provides a method of reducing or depleting the number of T regulatory cells in a tumor in a subject having cancer, the method comprising administering to the subject an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the antibody is administered in an amount or frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

In another aspect, the application provides a method of increasing IL-2 and/or IFN- γ production in T cells in a subject having cancer, the method comprising administering to the subject an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the antibody is administered in an amount or frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

In another aspect, the application provides a method of stimulating an immune response in a subject having cancer, the method comprising administering to the subject an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the antibody is administered in an amount or at a frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

In another aspect, the application provides a method of inhibiting the growth of tumor cells in a subject having cancer, the method comprising administering to the subject an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the antibody is administered in an amount or at a frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

In another aspect, the application provides a method of selecting an effective dose of a therapeutic agonistic antibody that specifically binds an immunostimulatory receptor or a schedule of antibody administration for treating a subject having cancer, the method comprising

(a) Administering the agonistic antibody to an animal model;

(b) obtaining a sample from the animal model;

(c) determining a receptor occupancy or receptor occupancy range for the agonistic antibody in the sample;

(d) using the receptor occupancy or receptor occupancy range obtained from step (c) to predict or predict an expected receptor occupancy or receptor occupancy range in the subject; and

(e) selecting an antibody or an administration dosage schedule sufficient to achieve and/or maintain a receptor occupancy of less than about 80% in the subject based on the expected receptor occupancy obtained in step (d).

In another aspect of the invention, the application provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a therapeutic agonistic antibody that specifically binds to an immunostimulatory receptor, or a pharmaceutical composition comprising the same, wherein the amount of agonistic antibody to be administered has been selected according to the methods described herein.

In another aspect, the present application provides a method of monitoring the level of a therapeutic agonistic antibody that specifically binds to an immunostimulatory receptor in a subject undergoing treatment for cancer, the method comprising

(a) Obtaining a sample from a subject;

(b) determining the receptor occupancy of said agonistic antibody in the sample;

(c) decreasing the amount or frequency of administration of the agonistic antibody to the subject if the receptor occupancy is greater than about 80% (e.g., 70%, 60%, 50%), or increasing the amount or frequency of the antibody if the receptor occupancy is less than about 20% (e.g., 30%, 40%, 50%, or 60%).

(d) Optionally repeating steps (a) - (c) until a receptor occupancy of about 20% to about 80% (e.g., about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%) is achieved and/or maintained.

In another aspect, the present application provides a method of treating cancer, the method comprising administering to a subject in need thereof an agonistic antibody that specifically binds to an immunostimulatory receptor and an additional therapy, wherein the additional therapy is administered at a fixed frequency and the agonistic antibody is administered at a dose and frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

In another aspect, the application provides a method of determining the effectiveness of a cancer treatment in a subject administered a therapeutic agonist antibody that specifically binds to an immunostimulatory receptor, the method comprising measuring the level of soluble OX40 in the subject (e.g., in a sample from the subject).

In some embodiments of the methods disclosed herein, the agonistic antibody is administered at a dose or frequency sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%. In certain embodiments, the receptor occupancy and/or receptor occupancy range is measured on day 1 after cycle 1 of the treatment regimen.

In certain embodiments of the methods disclosed herein, the agonistic antibody binds to an immunostimulatory receptor, e.g., a co-stimulatory receptor. In certain embodiments, the antibody binds to a member of the tumor necrosis factor receptor superfamily, ICOS, LFA-1(CD11a/CD18), CD2, CD7, CD30, CD40, CD54, CD160, BAFFR, HVEM, LIGHT, NKG2C, SLAMF7, and NKp 80. In one embodiment, the agonist antibody binds to OX40.

In certain embodiments of the methods disclosed herein, the agonistic antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or variants thereof. In some embodiments, the agonistic antibody comprises an Fc with enhanced binding to an activated fcyr. In some embodiments, the agonistic antibody is a human, humanized, or chimeric antibody. In some embodiments, the agonistic antibody is a bispecific antibody.

In some embodiments of the methods disclosed herein, the cancer to be treated is selected from the group consisting of: bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumor, lymphoma, leukemia, myeloma, sarcoma, non-small cell lung cancer, and virus-associated cancer. In some embodiments, the cancer is metastatic, refractory, or recurrent.

In some embodiments of the methods described herein, the subject (e.g., a human patient) is further administered one or more additional therapies (e.g., anti-PD 1 antibody, anti-PDL 1 antibody, anti-LAG 3 antibody, anti-CTLA 4 antibody, anti-TGF β antibody). The one or more additional therapies may be administered before, after, or concurrently with the agonistic antibody.

In some embodiments, the agonistic antibody, and optionally one or more additional therapies, are formulated into a pharmaceutical composition.

Brief description of the drawings

FIGS. 1A and 1B are tumor growth curves of CT26 mouse tumor models treated with indicated doses (unit: mg/kg) of IgG along with ligand-blocking OX40.23 (FIG. 1A) and ligand-non-blocking OX40.23 (FIG. 1B). 1

FIGS. 2A and 2B show mean and median tumor volumes, respectively, for the indicated doses (unit: mg/kg) of OX40.3 treated mice. FIGS. 2C and 2D show mean and median tumor volumes, respectively, for the indicated doses (unit: mg/kg) of OX40.23 treated mice.

FIG. 3A is a series of tumor growth curves for a CT26 mouse tumor model treated with a combination of OX40.23+ anti-PD 1 antibodies. Figure 3B is a graph showing the percent tumor growth inhibition of this combination treatment at different doses of OX40.23 antibody.

FIG. 4 is a series of tumor growth curves for a CT26 mouse tumor model treated with a combination of OX40.3+ anti-PD 1 antibodies.

FIG. 5 is a series of tumor growth curves for a CT26 mouse tumor model after treatment with indicated concentrations of OX40.23 at days 5 and 12 after tumor implantation, and concurrent or sequential (delayed) treatment with anti-PD-1 antibody. For simultaneous administration, anti-PD 1 antibody was administered on days 5,9, and 13. For delayed administration, the anti-PD 1 antibody was administered on days 10, 14, and 18.

Figure 6A is a schematic of the administration and sampling schedule of OX40.23 as monotherapy or in combination with an anti-PD 1 antibody. FIG. 6B is a graph of OX40 Receptor Occupancy (RO) in blood and tumor samples from mice treated with IgG, PD1, OX40.23(0.3, 3, and 10mg/kg), anti-PD 1 antibody + OX40.23(0.03, 0.3, 3, and 10 mg/kg). Figure 6C is a graph of total and occupied levels of OX40 receptors in day 8 and day 13 tumors. FIG. 6D is a graph showing percent tumor growth inhibition as a function of OX40 RO.

Figure 7A is a series of graphs showing% OX40RO in peripheral CD4+ T cells and Treg cells of human patients administered 20mg, 40mg, 80mg, 160mg, and 320mg OX 40.21. RO was measured at C1D1, C1D8 and C2D1 for a 2-week period. At 20mg, the observed peripheral OX40RO in CD4+ Tregs was 80%, saturated at doses ≧ 40 mg. FIG. 7B is a graph showing OX40.21 exposure at doses of OX40.21 from 20mg to 320mg in combination with nivolumab (nivolumab).

Figure 8A is a population pk (ppk) model used to describe the observed OX40.21 concentration data. FIG. 8B is a mathematical PK-PD model used to describe the relationship of drug concentration (Cmin 1: trough concentration after 1 dose) from human patients receiving OX40.21 treatment to peripheral blood RO on CD4+ T cells at C2D1, with the median number of RO (line segment) and 95% confidence interval (shaded area).

Figure 9 shows the predicted values of blood RO on CD4+ T cells (median and 90% prediction interval) at different OX40.21 doses and frequencies.

Fig. 10A and 10B show predicted human tumor RO at different dose regimens. Figure 10A provides the average tumor penetration and average target load. Figure 10B provides low tumor penetration and high target loading.

FIG. 11A is a graph showing the effect of different concentrations of OX40.21 on receptor occupancy (OX40.21 (binding)) and on OX40 surface expression (OX40.21 (total)). FIG. 11B is a graph summarizing the effect of% OX40RO on the total surface expression of OX40.

Fig. 12A and 12B are graphs showing the time course of OX40 surface expression at different dates ( days 1, 2,3, and 4) for isotype antibody and OX40.21 antibody at the indicated concentrations, respectively.

FIGS. 13A and 13B are graphs showing the effect of OX40.21 (FIG. 13A) or CD28 (FIG. 13B) concentration on total surface OX40 expression.

FIG. 14 is a graph showing T cell activation (reflected by IFN-. gamma.levels) as a function of OX40RO (y-axis) and OX40.21 concentration (x-axis).

FIG. 15A is a graph showing T cell proliferation (reflected by 3[ H ] -thymidine incorporation) as a function of OX40RO (y-axis) and OX40.21 concentration (x-axis). Fig. 15B and 15C are graphs showing the number of CD25+ T cells and the number of proliferating T cells, respectively, as a measure of% OX40 RO. .

Figure 16 is a schematic of an ELISA for measuring soluble OX40(sOX40) levels.

FIG. 17 is a graph showing the level of OX40 expression at the cell surface when cells were treated with the indicated concentrations of OX 40.21.

Figure 18 is a graph showing the level of soluble OX40 when cells were treated with the indicated concentrations of OX 40.21.

Figure 19 is a series of graphs showing fold increase of sOX40.21 relative to C1D1 when human patients are treated with OX40.21 monotherapy or OX40.21+ nivolumab in combination, wherein nivolumab is administered at 20mg, 40mg, 80mg, 160mg, and 320 mg.

Figure 20A is a graph showing the total levels of sOX40 on days 1, 2,3, and 4 when cells are treated with an isotype antibody, OX40.21, or an anti-CD 28 antibody. Figure 20B is a graph showing the levels of binding sOX40 at days 1, 2,3, and 4 when cells are treated with isotype antibody, OX40.21, or anti-CD 28 antibody.

FIG. 21A is a graph showing additive effects on sOX40 levels when cells are co-stimulated with OX40.21 and an anti-CD 28 antibody. FIG. 21B is a graph showing the effect on day 4 sOX40 levels after addition of OX40.21 to CD28 on day 3 of culture.

FIG. 22 is a schematic diagram showing an OX40 internalization assay.

Figure 23 is a schematic showing the internalization of OX40 in Tregs following activation of these Tregs with CD3/CD28, followed by treatment with IgG1, DT, OX40.21 (ligand blocking antibody) or OX40.8 (ligand non-blocking antibody).

Figure 24 is a series of graphs showing the effect of Fc γ R mediated cross-linking on the agonistic activity of OX 40.21.

Figure 25 is a schematic of dosing and sampling schedules for treatment of CT26 mouse tumor models with OX40.23 as monotherapy or in combination with an anti-PD 1 antibody for peripheral pharmacodynamic marker evaluation.

Fig. 26A is a series of graphs showing the effect of OX40.23 monotherapy or combination therapy with an anti-PD 1 antibody on ICOS, Ki67, and CD44 levels in CD4+ and CD8+ T cells. In the OX40 monotherapy panel, the X-axis labels are IgG, 90mpk, 30mpk, 10mpk, 3mpk, 1mpk, 0.3mpk, 0.1mpk, and 0.03mpk, in that order from left to right. In the panels of the OX40/PD-1 combinations, the labels on the X-axis are, from left to right, IgG, PD1, PD1+90mpk, PD1+30mpk, PD1+10mpk, PD1+3mpk, PD1+1mpk, PD1+0.3mpk, PD1+0.1mpk, PD1+0.03mpk, and PD1+0.01mpk, respectively. Figure 26B shows the percentage of CD8+ cells as Ki67+ in tumor stroma of human patients treated with OX40.21(20, 40, 80, 160, or 320mg) + nivolumab in combination. Figure 26C shows the percentage of FOXP3+ cells in tumor stroma of human patients treated with OX40.21(20, 40, 80, 160, or 320mg) + nivolumab. Figure 26D shows an immunohistochemical analysis of Ki67+ CD8+ T cells in tumor samples from human endometrial cancer patients treated with OX40.21(320mg) + nivolumab (240 mg). Figure 26E shows immunohistochemical analysis of FOXP3+ cells in human patient tumor samples from ovarian serous carcinoma (upper panel) and ovarian adenocarcinoma (lower panel).

FIGS. 27A and 27B are based on non-responders (tumor volume)>100mm³) Or responders (tumor volume ≤ 100 mm)³) Status, graph of the percentage of CD44 positive (fig. 27A) and Ki67 positive (fig. 27B) in CD8+ T cells. Figure 27C is a graph showing the absolute change in% Ki67+ CD8+ T cells calculated as anti-tumor activity (percentage of maximum decrease in tumor burden). Figure 27D is a graph showing changes (percent reduction in tumor burden) in Ki67+ CD8+ T cells by anti-tumor activity. PR: partial remission, PD: disease progression, SD: the disease is stable.

Figure 28 is a series of graphs showing the effect of increasing doses of anti-ICOS antibody in anti-ICOS + anti-PD 1 combination therapy on tumor growth inhibition in a mouse model.

Figure 29 is a series of graphs showing the exposure-response relationship of mIgG1 and mIgG2a monoclonal anti-OX 40 antibodies in a mouse MC38 tumor model.

FIG. 30 shows the mechanism of action of OX-40 agonist antibodies (BMS-986178 instead of mouse antibodies) on OX-40. Fc γ R ═ Fc γ receptors; FoxP 3-forkhead box protein P3; NK ═ natural killer cells; BMS-986178: an OX40 monoclonal antibody.

Figure 31 shows T cell activation measured by geometric mean fluorescence intensity of CD25 on CD4+ T cells treated with OX-40 agonist antibody (BMS-986178 in place of mouse antibody) or isotype antibody [ GMFI ].

Figures 32A and 32B are graphs summarizing the effect of% OX40RO on the total surface expression of OX40 compared to isotype antibody control after treatment of CHO cells with either CD137 monoclonal antibody (figure 32A) or CD28 monoclonal antibody (figure 32B).

FIGS. 33A-33I show the hook effect and dose dependence in the Treg suppression assay when CHO cells were treated with OX-40 agonist antibodies. Figure 33A is a schematic of this Treg inhibition assay. Figure 33B shows IL-2 expression after treatment with different concentrations of BMS-986178(BMS-986178 instead of mouse antibody) in CD4+ in the presence of tregs. FIG. 33C shows IL-2 expression in CD4+ after treatment with various concentrations of BMS-986178(BMS-986178 instead of mouse antibody) in the absence of Tregs. Figure 33D shows OX40 expression on Tregs in corresponding cultures after treatment with different concentrations of BMS-986178(BMS-986178 substituted for mouse antibody) in CD4+ in the presence of Tregs. Figure 33E shows OX40 expression on Tregs in corresponding cultures after treatment with different concentrations of BMS-986178(BMS-986178 substituted for mouse antibody) in CD4+ in the absence of Tregs. FIG. 33F shows IL-2 expression in CD8+ after treatment with various concentrations of BMS-986178(BMS-986178 instead of mouse antibody) in the presence of Tregs. FIG. 33G shows IL-2 expression in CD8+ after treatment with various concentrations of BMS-986178(BMS-986178 substituted for mouse antibody) in the absence of Tregs. Figure 33H shows OX40 expression on Tregs in corresponding cultures after treatment with different concentrations of BMS-986178(BMS-986178 instead of mouse antibody) in CD8+ in the presence of Tregs. Figure 33I shows OX40 expression on Tregs in corresponding cultures after treatment with various concentrations of BMS-986178(BMS-986178 in place of mouse antibody) in CD8+ in the absence of Tregs.

FIG. 34A is the expression of OX40RO on tumor Tregs 7 days after treatment with 0.03mg/kg, 0.3mg/kg, 3.0mg/kg, 10mg/kg of BMS-986178(BMS-986178 instead of mouse antibody) or control IgG. FIG. 34B is total OX40RO expression measured in mean fluorescence intensity after 7 days of treatment with 0.03mg/kg, 0.3mg/kg, 3.0mg/kg, 10mg/kg BMS-986178(BMS-986178 instead of mouse antibody) or control IgG. FIG. 34C is the expression of OX40RO on tumor Tregs at 10, 17 and 22 days after treatment with 0.5mg/kg BMS-986178(BMS-986178 instead of mouse antibody), 5mg/kg BMS-986178(BMS-986178 instead of mouse antibody) or control PD-1 monoclonal antibody. FIG. 34D is total OX40RO expression determined on tumor Tregs as measured by mean fluorescence intensity at 10, 17, and 22 days after treatment with 0.5mg/kg BMS-986178(BMS-986178 instead of mouse antibody), 5mg/kg BMS-986178(BMS-986178 instead of mouse antibody), or control PD-1 monoclonal antibody.

Figure 35A is a graph showing the level of soluble OX40 when cells are treated with labeled concentrations of CD28 monoclonal antibody or an isotype antibody control. FIG. 35B is a graph showing the levels of total soluble OX40 and drug-bound soluble OX40 when cells were treated with indicated concentrations of BMS-986178(BMS-986178 instead of mouse antibody).

FIG. 36 is a schematic representation of internalization of BMS-986178(BMS-986178 in place of mouse antibody) that binds to OX40 when Tregs and CD4+ T cells are treated with 0.01nM and 100nM BMS-986178(BMS-986178 in place of mouse antibody).

FIG. 37 is a schematic model of the relationship between BMS-986178(BMS-986178 in place of mouse antibody) dose, OX40RO, OX40 expression and PD modulation.

FIG. 38 shows tumor volume and number of tumor-free mice in CT26 mouse tumor models treated with control monoclonal antibody (mIgG1), BMS-986178(BMS-986178 instead of mouse antibody), anti-PD-1, anti-CTLA-4, BMS-986178(BMS-986178 instead of mouse antibody) in combination with anti-PD-1, or BMS-986178(BMS-986178 instead of mouse antibody) in combination with anti-CTLA-4. mIgG ═ mouse immunoglobulin G; TF is tumor-free; anti-PD-1-4H 2 mIgG 1D 265A and anti-CTLA-4-9D 9 mIgG2 b. Tumor volumes ± standard deviations were measured twice weekly starting on day 6 post-implantation (start of treatment).

Figure 39 shows the study design of monotherapy dose escalation (BMS-986178 only) compared to combination therapy (BMS-986178 in combination with either nivolumab or ipilimumab). DLT-dose-limiting toxicity; ECOG PS ═ state of performance of Eastern bank cancer clinical research Cooperative organization (Eastern Cooperative Oncology Group); IV, intravenous injection; MTD-maximum tolerated dose; Q2W once every 2 weeks; Q3W once every 3 weeks; RECIST is a response evaluation criterion for solid tumors; RP 2D-recommended phase 2 dose. FIG. 40 shows the pharmacokinetics of BMS-986178+ -nivolumab or ipilimumab.

FIG. 41 is peripheral OX40RO on peripheral Tregs treated with 20mg, 40mg, 80mg, 160mg, or 320mg of OX-40.21, using surface markers C1D1, C1D8, C2D1, or C5D 1.

FIG. 42 is total OX40 expression on peripheral Tregs measured by mean fluorescence intensity after treatment with 20mg, 40mg, 80mg, 160mg, or 320mg of OX-40.21 using surface markers C1D1, C1D8, C2D1, or C5D 1.

Figure 43A shows the fold change over time for sOX40 in individual patient samples at different doses. Fig. 43B shows the normalized AUC of sOX40 at different doses. sOX40 normalized AUC adopted (AUC 0-T)_{Finally, the}/T_{Finally, the}) To account for patients at different follow-up times; AUC0-T_{Finally, the}Area under the curve from time 0 to last visit; t is_{Finally, the}Last visit time.

FIGS. 44A and 44B show that BMS-986178+ -nivolumab or ipilimumab stimulates the production of IFN- γ (FIG. 44A) and IP-10 (FIG. 44B).

FIGS. 45A and 45B show that BMS-986178+ -nivolumab or ipilimumab increased the level of proliferative (Ki67+) CD4+ (FIG. 45A) and CD8+ (FIG. 45B) effector memory T cells.

Figures 46A to 46H show validation results of a human total soluble OX40 biomarker assay for determining total soluble OX40 levels in human serum. FIG. 46A shows a calibration curve between BMS-986178 and two different OX40 proteins (OX40-His _ Sino and OX40-Fc _ R & D). Figure 46B shows the correlation of serum OX40 between two antibody pairs. Fig. 46C and 46D show the correlation between OX40 antibody pairs in serum. Fig. 46C and 46D show dilution parallelism and linearity, respectively. Fig. 46E shows the selectivity of BMS-986178. Fig. 46F and 46G show drug interference data. Fig. 46H shows the storage and freeze-thaw stability of BMS-986178 under 5 different conditions.

Figure 47 shows soluble OX40 levels determined using a human total soluble OX40 biomarker assay in normal healthy humans and three different cancer subjects (head and neck, ovarian, and cervical cancer).

Figure 48 shows drug interference data for OX40.8 antibody.

Detailed description of the invention

Provided herein are methods of enhancing an immune response using an agonistic antibody that specifically binds to an immunostimulatory receptor, administered alone or in combination with other immunostimulatory agents and/or cancer therapies, in an amount sufficient to achieve and/or maintain a receptor occupancy of less than about 80%. The methods described herein can be used in a variety of oncology applications, for example, treating cancer or inhibiting tumor growth.

Definition of

In order that the following description may be more readily understood, certain terms are first defined. Further definitions are given throughout the description.

As used herein, "immunostimulatory receptor" refers to a receptor involved in stimulating an immune response. Such receptors include, for example, co-stimulatory receptors.

As used herein, "co-stimulatory receptor" refers to a receptor that transmits a co-stimulatory signal to an immune cell. Examples of co-stimulatory receptors include, but are not limited to: members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), ICOS (CD278), CD28, LIGHT, CD40L, TIM1, SLAM, CD1, CD2, CD226, LFA-1(CD11A, CD18), CD2, CD5, CD7, CD30, CD54, CD97, CD154, CD160, LIGHT, NKG2C, SLAMF7 and NKp 80.

As used herein, "agonistic antibody" refers to an antibody that is an agonist of an immunostimulatory receptor, such as antibodies that: it is capable of stimulating the activity of a protein which in turn stimulates immune cells, thereby enhancing the immune system (or immune response) of a subject.

As used herein, "an agonistic antibody that binds to an immunostimulatory receptor," or synonymously expressed, refers to an antibody that specifically binds to an immunostimulatory receptor (e.g., a co-stimulatory receptor such as a member of the tumor necrosis factor receptor superfamily) and activates the receptor and/or its downstream signaling pathways.

As used herein, "tumor necrosis factor receptor superfamily" or "TNFRSF" refers to a protein superfamily of cytokine receptors that have a cysteine-rich domain in their extracellular domain that binds to a natural ligand; this superfamily includes TNFR1, TNFR2, HVEM, LT β R, OX40, CD27, CD40, FAS, DCR3, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, OPG, RANK, FN14, TACI, BAFFR, BCMA, GITR, TROY, DR3 (death receptor 3), DR6 (death receptor 6), XEDAR (epidermal cell proliferator A2 receptor) and NGFR (see, e.g., Croft et al, Nat Rev Drug Discov 2013; 12: 147-.

As used herein, the term "OX40" refers to a receptor that is a member of TNFRSF that binds to OX40 ligand (OX 40-L). OX40 is also known as tumor necrosis factor receptor superfamily member 4(TNFRSF4), ACT35, IMD16, TXGP1L, and CD 134. The term "OX40" includes any variant or isomer of OX40 that is naturally expressed by a cell.

The amino acid sequence of human OX40 precursor (accession NP-003318.1) is set forth in SEQ ID NO: 1. The amino acid sequence of the extracellular domain of mature human OX40 is set forth in SEQ ID NO 2. The amino acid sequence of cynomolgus monkey OX40 is set forth in SEQ ID NO. 3. The amino acid sequence of human OX40-L is set forth in SEQ ID NO. 4.

The terms of Programmed Death Protein 1("Programmed Death1", "Programmed Cell Death1", "Programmed Death1", "Programmed Cell Death 1"), Protein PD-1("Protein PD-1"), "PD-1", "PD1", "PD1", "PDCD1", "hPD-1", "hPD-I" and the like refer to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes human PD-1(hPD-1), variants, isomers, and species homologs of hPD-1, as well as analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found by GenBank accession No. U64863.

"programmed death protein ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), and upon binding to PD-1, downregulates T cell activation and cytokine secretion. The term "PD-L1" as used herein includes variants, isomers and species homologs of human PD-L1(hPD-L1), hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete sequence of hPD-L1 can be found by GenBank accession No. Q9NZQ 7.

The terms "cytotoxic T lymphocyte-associated antigen-4", "CTLA-4 antigen" and "CD152" (see, e.g., Murata (1999) am. J. Pathol.155: 453-plus 460) are used interchangeably and include variants, isomers, species homologs of human CTLA-4, and analogs having at least one epitope in common with CTLA-4 (see, e.g., Balzano (1992) int. J. cancer suppl.7: 28-32.) the complete sequence of human CTLA-4 is set forth in GenBank accession number Ll 5006.

The term "antibody" as used herein may include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. In one embodiment, an "antibody" refers to a glycoprotein, or antigen-binding portion thereof, interconnected by at least two heavy (H) chains and two light (L) chains via disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V)_H) And a heavy chain constant region. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region consists of three domains, CH1, CH2 and CH 3. In some naturally occurring antibodies, each light chain includes a light chain variable region (abbreviated herein as V)_L) And a light chain constant region. The light chain constant region consists of one domain of CL. V_HAnd V_LRegions can be further subdivided into regions of high variability, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

Antibodies typically bind specifically with high affinity to homologous antigens, as reflected in the dissociation constant (K)_D) Is 10^-7To 10^-11M or less. Any greater than about 10^-6K of M_DGenerally considered to represent non-specific binding. As used hereinAn antibody that "specifically binds" to an antigen refers to an antibody that binds with high affinity to the antigen and to substantially the same antigen-this means that K is_DIs 10^-7M or less, more preferably 10^-8M or less, even more preferably 5 × 10^-9M or less, most preferably 10^-8M to 10^-10M or lower-but not with high affinity to an unrelated antigen. An antigen is "substantially identical" to a given antigen if it is highly identical to the sequence of the given antigen, e.g., if the antigen exhibits at least 80%, at least 90%, more preferably at least 95%, more preferably at least 97%, even more preferably at least 99% sequence identity to the sequence of the given antigen. For example, an antibody that specifically binds human OX40 can cross-react with OX40 from certain non-human primates (e.g., cynomolgus monkeys), but not with OX40 from other species (e.g., murine OX40) nor with antigens other than OX40.

The immunoglobulin may be from any one of the common isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. In some species, IgG isotypes can be subdivided into subclasses: there are IgG1, IgG2, IgG3 and IgG4 in humans and IgG1, IgG2a, IgG2b and IgG3 in mice. In certain embodiments, the anti-OX 40 antibody described herein is an IgG1 or IgG2 isotype. Immunoglobulins, such as IgG1, exist in a number of allotypes that differ from each other by at most a few amino acids. "antibody" can include, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric antibodies and humanized antibodies; human and non-human antibodies; fully synthesizing an antibody; and single chain antibodies.

As used herein, the term "antigen-binding portion" or "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., human OX 40). It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion of an antibody" include: (i) fab fragment from V_L、V_HCL anda monovalent fragment consisting of the CH1 domain which consists of a VH domain; (ii) f (ab')₂A fragment which is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) fd fragment consisting of V_HAnd a CH1 domain; (iv) fv fragment consisting of V of a single arm of an antibody_LAnd V_HDomain composition; (v) dAb fragments (Ward et al, (1989) Nature 341:544-546) from V_HDomain composition; and (vi) an isolated Complementarity Determining Region (CDR); or (vii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains V of the Fv fragment_LAnd V_HEncoded by different genes, but which can be joined by recombinant means via a synthetic linker so that they are produced as a single protein chain, where V_LAnd V_HThe regions pair to form monovalent molecules (known as single chain fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These and other possible constructs are described in Chan&Carter (2010) nat. rev. immunol.10: 301. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility in the same manner as are intact antibodies. Antigen binding portions may be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

A "bispecific antibody" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or ligation of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.Exp. Immunol.79: 315-; kostelny et al, J.Immunol.148,1547-1553 (1992).

The term "monoclonal antibody" as used herein refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope, or to a composition of antibodies in which all antibodies exhibit a single binding specificity and affinity for a particular epitope. Typically, such monoclonal antibodies are derived from a single cell or nucleic acid encoding the antibody and are amplified without the deliberate introduction of any sequence changes. Accordingly, the term "human monoclonal antibody" refers to a monoclonal antibody having variable regions (and, optionally, constant regions) derived from human immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma, e.g., obtained by fusing a B cell obtained from a transgenic or transfectant non-human animal (e.g., a transgenic mouse having a genome comprising a human heavy chain transgene and a light chain) with an immortalized cell.

As used herein, the term "recombinant human antibody" includes all human antibodies that are prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for human immunoglobulin genes, or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibodies, e.g., from transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies comprise variable and constant regions utilizing specific human germline immunoglobulin sequences: the sequences are encoded by germline genes but include subsequent rearrangements and mutations, such as those that occur during antibody maturation. As is known in the art (see, e.g., Lonberg (2005) Nature Biotech.23 (9): 1117-1125), the variable region comprises an antigen-binding domain encoded by a plurality of different genes that are rearranged to form antibodies specific for foreign antigens. In addition to rearrangement, the variable region may be further modified by multiple single amino acid changes (known as somatic mutations or hypermutations) to increase the affinity of the antibody for foreign antigens. The constant region will change in further response to the antigen (i.e., isotype switching). Thus, the nucleic acid sequences encoding the light and heavy immunoglobulin polypeptides are not identical, but are substantially identical or similar (i.e., at least 80% identical) to the original germline sequences, subject to rearrangement and somatic mutation in response to the antigen.

"human" antibody (HuMAb) refers to an antibody having variable regions that: both the framework and CDR regions in the variable regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The antibodies described herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include such antibodies: wherein CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are synonymous.

"humanized" antibodies are antibodies in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside of the CDR domains have been replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. "humanized" antibodies retain antigen specificity similar to the original antibody.

"chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, for example, an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

As used herein, "isotype" refers to the class of antibodies encoded by heavy chain constant region genes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody specifically binding to an antigen".

As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities.

As used herein, an antibody that "inhibits the binding of OX40-L to OX40" is intended to refer to an antibody that inhibits the binding of OX40-L to OX40.

"Effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or the biochemical event that results from such an interaction. Exemplary "effector functions" include: c1q binding, Complement Dependent Cytotoxicity (CDC), Fc receptor binding, Fc γ R mediated effector functions such as ADCC and antibody dependent cell mediated phagocytosis (ADCP), and down-regulation of cell surface receptors (e.g., B cell receptors; BCR). Such effector functions typically require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).

An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcR binding to IgG antibodies include receptors of the Fc γ R family, including allelic variants and alternatively spliced forms of these receptors. The Fc γ R family consists of three activating receptors (Fc γ RI, Fc γ RIII and Fc γ RIV in mice; Fc γ RIA, Fc γ RIIA and Fc γ RIIIA in humans) and one inhibiting receptor (Fc γ RIIB). Table 1 summarizes various properties of human Fc γ R. Most innate effector cell types co-express one or more activating Fc γ rs and inhibitory Fc γ RIIB, while Natural Killer (NK) cells selectively express one activating Fc receptor (Fc γ RIII in mice and Fc γ RIIIA in humans) and do not express inhibitory Fc γ RIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the type of activating Fc receptor to which it binds.

Table 1: properties of human Fc. gamma.R

"Fc region" (fragment crystalline region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component of the classical complement system (C1 q). Thus, the Fc region includes a region of the constant region of an antibody other than the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fc region comprises the C on each of the two heavy chains of an antibody_H2And C_H3A constant domain; the Fc region of IgM and IgE consists of three heavy chain constant domains (C) per polypeptide chain_HDomains 2-4). For IgG, the Fc region includes the immunoglobulin domains C γ 2 and C γ 3, and the hinge between C γ 1 and C γ 2. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position C226 or P230 (or an amino acid between these two amino acids) to the carboxy-terminus of the heavy chain, with numbering according to the EU index of Kabat. Kabat et al (1991) Sequences of Proteins of Immunological Interest, National Institutes of health, Bethesda, Md; see also FIGS. 3C-3F of U.S. application publication No. 2008/0248028. C of human IgG Fc region_H2The domain extends from about amino acid 231 to about amino acid 340, and C_H3The domain is located at C in the Fc region_H2The C-terminal side of the domain, i.e., it extends from around amino acid 341 to around amino acid 447 of the IgG. As used herein, an Fc region can be a native sequence Fc, including any allotropic variant, or a variant Fc (e.g., a non-naturally occurring Fc). Fc may also refer to this region in isolation, or in the context of a protein polypeptide comprising Fc, such as "binding protein comprising an Fc region," also known as an "Fc fusion protein" (e.g., an antibody or immunoadhesin).

As used herein, the terms "specific binding," "selective binding," and "specific binding" refer to an antibody that binds to an epitope on a predetermined antigen, but does not bind to other antigens. Typically, the antibody (I) is as in, for example, a BIACORE 2000 Surface Plasmon Resonance (SPR) instrument, toEquilibrium dissociation constant (K) of binding when predetermined antigen as analyte and antibody as ligand are measured using Surface Plasmon Resonance (SPR) technique_D) Less than about 10^-7M, e.g. less than about 10^-8M、10^-9M or 10^-10M, or even lower; and (ii) binds to the predetermined antigen with at least 2-fold greater affinity than it binds to a non-specific antigen other than the predetermined antigen (e.g., BSA, casein) or to the predetermined antigen or closely related antigens.

The term "k_assoc"or" k_a", as used herein, is intended to refer to the binding rate constant for a particular antibody-antigen interaction, whereas the term" k "is used_dis"or" k_d", as used herein, is intended to refer to the off-rate constant for a particular antibody-antigen interaction. The term "K" as used herein_D"means the equilibrium dissociation constant, which is measured from k_dAnd k is_aRatio of (i.e. k)_d/k_a) Obtained and expressed in molar concentration (M). K of antibody_DThe values may be determined using methods established in the art. Determination of K of antibodies_DA preferred method of value is the use of surface plasmon resonance, preferably using a biosensor system, e.g.

SPR system or flow cytometer and Scatchard analysis.

As used herein, the term "high affinity" IgG antibody refers to K against a target antigen_DIs 10^-8M is less, or more preferably 10^-9M is less, or more preferably 10^-10M or lower. However, the "high affinity" binding may differ for antibodies of other antibody isotypes. For example, for IgM isotype, "high affinity" binding means having 10^-7M or less, more preferably 10^-8K of M or less_DThe antibody of (1).

The term "EC50," in the context of in vitro or in vivo assays using antibodies, refers to the concentration of antibody at which a response of 50% of the maximal response is induced, i.e., the half-way between the maximal response and baseline.

"receptor occupancy" or "occupancy of a receptor," as used herein, refers to the amount of agonistic antibody that binds to an immunostimulatory receptor. "% receptor occupancy" or "% occupancy of receptors" can be calculated using the following formula: ([ Δ MFI for the test ]/[ total Δ MFI ]) × 100. Δ MFI is calculated as follows: the background stained MFI of isotype control antibody was subtracted from the Mean Fluorescence Intensity (MFI) of bound agonistic antibody. The total receptor level was determined as follows: a saturating amount of agonistic antibody is added to determine the maximum expression of a particular immunostimulatory receptor and thus its MFI. Another method of calculating total receptor expression is to use antibodies directed to the same immunostimulatory receptor but not competing with agonistic antibodies that calculate receptor occupancy.

An "immune response" refers to a biological response in a vertebrate to a foreign agent that protects the body from these agents and the diseases caused by them. The immune response is a function produced by certain cells in the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) as well as by any of these cells or soluble macromolecules produced by the liver, including antibodies, cytokines, and complements), resulting in the selective targeting, binding, damage, destruction, and/or clearance from the vertebrate of invading pathogens, pathogen-infected cells or tissues, cancerous cells or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues. Immune responses include, for example, activation or suppression of T cells, e.g., effector T cells or Th cells, e.g., CD4+ or CD8+ T cells, or suppression of Treg cells.

"immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of contracting a disease or the recurrence of a disease by a method that includes inducing, enhancing, suppressing or otherwise altering the immune response.

"T Effect (T)_eff) "cell" refers to T cells (e.g., CD4+ and CD8+ T cells) and T helper cells (Th cells) that have cytolytic activity, secrete cytokines and activate and direct other immunityCells, but not regulatory T cells (Treg cells).

The enhanced ability to stimulate the immune response or immune system may be the result of enhanced agonist activity of the T cell co-stimulatory receptor and/or enhanced antagonist activity of the inhibitory receptor. Enhancement of the ability to stimulate an immune response or immune system can be measured by EC in an assay for measuring an immune response₅₀Or a fold increase in the maximum activity level, such as an assay that measures changes in: cytokine or chemokine release, cytolytic activity (measured directly on target cells, or indirectly by detecting CD107a or granzyme), and proliferation. The ability to stimulate an immune response or immune system activity can be enhanced by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold, or more.

As used herein, "administering" refers to physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibodies described herein may be administered by a parenteral route, e.g., by a topical, epidermal or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual or topical. Administration may also be carried out, for example, once, multiple times, and/or over one or more extended periods of time.

As used herein, the term "T cell mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD 8)⁺Cells) and helper T cells(e.g., CD4⁺Cells), the mediated response. T cell-mediated responses include, for example, the cytotoxic effects and proliferation of T cells.

As used herein, the term "Cytotoxic T Lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are mainly mediated by CD8+ T cells.

As used herein, the terms "inhibit" or "block" (e.g., inhibit/block the binding of OX40-L to OX40 on a cell) are used interchangeably and encompass partial and complete inhibition/blocking. Likewise, a "blocking antibody" refers to an antibody that blocks the binding of a ligand to its receptor, e.g., OX40.21 inhibits the binding of OX40 to its ligand, and is therefore referred to as a blocking antibody. In contrast, antibodies that do not block ligand binding to their receptor, such as OX40.8, are referred to as "non-blocking antibodies".

As used herein, the term "inhibiting the growth of a tumor" includes any measurable reduction in the growth of a tumor, for example, inhibiting the growth of a tumor by at least about 10%, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%.

As used herein, "cancer" refers to a broad class of diseases characterized by uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the cells forming a malignant tumor that may invade adjacent tissues and may metastasize to distant parts of the body through the lymphatic system or blood.

The term "treatment" as used herein refers to any type of intervention or procedure performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, inhibiting, or slowing or preventing the progression, severity, or recurrence of the symptoms, complications, conditions, or biochemical indicators associated with the disease. "preventing" refers to administration to a subject without a disease to prevent the onset of the disease or to minimize its effect if the disease occurs.

"hematological malignancies" include lymphoma, leukemia, myeloma orLymphoid malignancies, and cancers of the spleen and lymph nodes. Exemplary lymphomas include B cell lymphoma (a B cell leukemia) and T cell lymphoma. B cell lymphomas include hodgkin lymphoma and most non-hodgkin lymphomas. Non-limiting examples of B cell lymphomas include diffuse large B cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma (overlapping chronic lymphocytic leukemia), Mantle Cell Lymphoma (MCL), Burkitt's lymphoma, mediastinal large B cell lymphoma, lymphomas,

Macroglobulinemia, nod-marginal zone B cell lymphoma, spleen-marginal zone lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphoid granuloma. Non-limiting examples of T cell lymphomas include extranodal T cell lymphomas, cutaneous T cell lymphomas, anaplastic large cell lymphomas, and angioimmunoblastic T cell lymphomas. Hematologic malignancies also include leukemias, such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and acute lymphocytic leukemia. Hematological malignancies further include myelomas such as, but not limited to, multiple myeloma and smoldering multiple myeloma. Other hematological and/or B-cell or T-cell related cancers are also encompassed within the term hematological malignancy.

The term "effective dose" or "sufficient dose" is defined as an amount of a drug (e.g., an agonistic antibody that binds an immunostimulatory receptor) sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent refers to any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression, which manifests itself in decreased severity of disease symptoms, increased frequency and duration of asymptomatic phases of the disease, or prevention of a disorder or disability resulting from the disease condition.

With respect to solid tumors, an effective amount includes an amount sufficient to cause tumor shrinkage and/or to reduce the growth rate of the tumor (e.g., inhibit tumor growth) or to prevent or delay the proliferation of other unwanted cells. In certain embodiments, an effective amount is an amount sufficient to delay tumorigenesis. In certain embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations. The effective amount of the medicament or composition may be: (i) reducing the number of cancer cells; (ii) reducing the size of the tumor; (iii) inhibit, hinder, and possibly prevent cancer cell infiltration into peripheral organs; (iv) inhibition, i.e., slowing and possibly preventing tumor metastasis to some extent; (v) inhibiting tumor growth; (vi) preventing or delaying the appearance and/or recurrence of a tumor; and/or (vii) relieve to some extent one or more symptoms associated with cancer.

As used herein, the terms "fixed dose", "constant dose" and "fixed constant dose" are used interchangeably and refer to a dose administered to a patient without regard to the patient's weight or Body Surface Area (BSA). Thus, a fixed or fixed dose is not provided in a mg/kg dose, but rather in the absolute amount of the formulation.

A "prophylactically effective amount" or "prophylactically effective dose" of a drug is a dose that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing or relapsing, inhibits the development or relapse of the disease. The ability of a therapeutic or prophylactic agent to promote regression of a disease or inhibit the occurrence or recurrence of a disease can be assessed using a variety of methods known to skilled practitioners, for example, by clinical trials in human subjects, using animal model systems that predict efficacy in humans, or by measuring the activity of the agent in vitro assays.

For example, an "anti-cancer agent" is a drug that slows the progression of cancer in a subject or promotes the regression of cancer in a subject. In a preferred embodiment, the therapeutically effective amount of the drug promotes regression of the cancer to the point of eliminating the cancer. By "promoting cancer regression," it is meant that an effective amount of a drug, administered alone or in combination with an anti-neoplastic agent, results in a reduction in the growth or size of a tumor, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of the asymptomatic phase of the disease, prevention of a disorder or disability due to the affliction with the disease, or otherwise ameliorates the disease symptoms in the patient. By "pharmacologically effective" is meant that the drug promotes cancer regression in the patient. By "physiologically safe" is meant that toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and/or body level are at an acceptably low level following administration of the drug.

As an example of treating a tumor, a therapeutically effective amount or dose of the drug preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and even more preferably by at least about 80%, relative to an untreated subject. In a most preferred embodiment, the therapeutically effective amount or dose of the drug completely inhibits cell growth or tumor growth, i.e., preferably inhibits cell growth or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be assessed using the detection methods described below. Alternatively, such a property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, such inhibition being measured in vitro according to assays known to those skilled in the art. In other preferred embodiments described herein, regression of the tumor may be observed and may last for at least about 20 days, more preferably at least about 40 days, even more preferably at least about 60 days.

The terms "patient" and "subject" refer to any human or non-human animal that is undergoing prophylactic or therapeutic treatment. For example, the methods and compositions described herein can be used to treat a subject or patient having cancer, e.g., an advanced solid tumor.

Use of an alternative term (e.g., "or") should be understood to refer to one, two, or any combination of the alternatives.

As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any listed or enumerated ingredient.

As used herein, the term "about," in the context of a numerical value or range, refers to ± 10% of the numerical value or range.

Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range described herein is to be understood as including the value of any integer within the range, and where appropriate, the fraction thereof (e.g., one tenth and one hundredth of an integer) unless otherwise indicated.

Various aspects described herein are described in further detail in the following sections.

I. Application method

The present application provides methods of treating cancer using agonistic antibodies that bind to immunostimulatory receptors in an amount (dose) or at a frequency (antibody administration schedule) sufficient to achieve and/or maintain non-saturated Receptor Occupancy (RO). As demonstrated in the examples, a saturating or near saturating dose of an agonistic antibody that binds to an immunostimulatory receptor (a dose that results in RO > 80%) results in a decrease in anti-tumor efficacy as compared to a non-saturating dose (a dose that results in RO less than about 80%), reflected in markers of T cell activation and proliferation. This suggests that treating a subject with cancer by administering a sub-saturating dose of an agonistic antibody that binds an immunostimulatory receptor may provide greater therapeutic benefit than administering a saturating dose.

Accordingly, the present application provides a method of treating cancer comprising administering to a subject in need thereof (e.g., a human patient) an agonistic antibody that binds an immunostimulatory receptor, wherein the amount or frequency of administration of the antibody is sufficient to achieve and/or maintain less than about 80% RO.

The present application also provides a method of treating cancer in a subject (e.g., a human patient) in which an amount or frequency of agonistic antibodies that bind to an immunostimulatory receptor sufficient to achieve and/or maintain RO of less than about 80% has been determined for the subject, the method comprising administering to the subject a sufficient amount of the antibody.

The present application also provides a method of reducing or depleting the number of T regulatory cells in a tumor in a subject having cancer, comprising administering to the subject an agonistic antibody that binds to an immunostimulatory receptor, wherein the antibody is administered in an amount or frequency sufficient to achieve and/or maintain less than about 80% RO.

The present application also provides a method of increasing IL-2 and/or IFN- γ production in T cells of a subject having cancer, comprising administering to the subject an agonistic antibody that binds an immunostimulatory receptor, wherein the antibody is administered in an amount or frequency sufficient to achieve and/or maintain less than about 80% RO.

The present application also provides a method of stimulating an immune response in a cancer patient, comprising administering to the patient an agonistic antibody that binds an immunostimulatory receptor, wherein the antibody is administered in an amount or frequency sufficient to achieve and/or maintain less than about 80% RO.

The present application also provides a method of inhibiting the growth of a tumor or tumor cell in a subject having cancer, the method comprising administering to the subject an agonistic antibody that binds an immunostimulatory receptor, wherein the antibody is administered in an amount or frequency sufficient to achieve and/or maintain less than about 80% RO.

In some embodiments, the agonistic antibody is administered in an amount or frequency sufficient to achieve and/or maintain less than about 70% RO, e.g., less than about 60%, less than about 50%, less than about 40%, or less than about 30% RO. In certain embodiments, the amount applied is sufficient to achieve and/or maintain a RO range of about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, about 60% to about 70%, about 30% to about 60%, or about 30% to about 50%.

In certain embodiments of the methods described herein, the agonistic antibody is administered in an amount or frequency sufficient to achieve and/or maintain less than about 20% RO, e.g., less than about 15%, less than about 10%, less than about 5%, or from about 5% to about 20%, from about 10% to about 20%, or from about 15% to about 20% RO. In some embodiments, less than 20% RO is achieved and/or maintained by intermittent or pulsed administration to a subject (e.g., a human patient). For example, pulsed administration may include combination therapy of an agonistic antibody and other agents, where the agonistic antibody is administered once every 8 or 12 weeks and the other agent (e.g., anti-PD 1 antibody) is administered once every 4 weeks.

In some embodiments, RO is measured on day 1 after cycle 1 in an antibody treatment regimen. In some embodiments, RO is measured in mid-cycle in an antibody treatment regimen. In some embodiments, RO is measured at the beginning of a certain period of an antibody treatment regimen. In some embodiments, RO is measured on multiple days in one or more cycles of the antibody treatment regimen. For example, in one embodiment, the RO is measured on days 1, 7, and/or 14 of a 14 day cycle, and thereafter at set intervals (e.g., every 2 weeks). In certain embodiments, RO is measured when the antibody concentration approaches Cmax, Cmin, and/or Ctrough, and/or at the peak of the induced immune response when expression of the immunostimulatory receptor is predicted to be highest (e.g., day 7-14 after administration).

Methods of measuring RO are well known in the art. For example, RO can be measured in a biological sample (e.g., blood) by flow cytometry using a fluorescently labeled antibody of interest, as described in example 7A. Methods for measuring receptor occupancy have been described, for example, in Liang et al, Cytometry B Clin Cytom 2016; 90:117-27, Ciccimura et al, Anal Chem 2017; 89: 5115-. RO from peripheral blood and in tissues can also be measured using affinity extraction liquid chromatography-mass spectrometry (AE LC-MS) by assessing the total level of agonistic antibodies of the immunostimulatory receptor of interest, the total level of immunostimulatory receptors, and the total level of complexes.

In some embodiments, RO binding to an agonistic antibody of an immunostimulatory receptor can be used to provide information for human dose selection. For example, the present application provides a method of treating cancer comprising (a) administering to a subject in need thereof an antibody that specifically binds to an immunostimulatory receptor, (b) measuring RO in a sample from the subject, and (c) determining the amount of antibody administered to the subject based on the measured RO and/or RO range, or determining a schedule of antibody administration based on the measured RO and/or RO range.

In some embodiments, the present application provides a method of selecting an effective dose of a therapeutic agonistic antibody that specifically binds an immunostimulatory receptor or a schedule of antibody administration for treating a subject having cancer, the method comprising

(a) Administering the antibody to a subject;

(b) obtaining a sample (e.g., blood) from a subject;

(c) determining RO on cells in the sample; and

(d) an amount of antibody or schedule of antibody administration sufficient to achieve and/or maintain less than about 80% RO or RO range (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, less than about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30%) is selected.

RO for agonistic antibodies can be initially determined in preclinical animal models to provide information for administration in other mammals, such as humans. For example, in some embodiments, the present application provides a method of selecting an effective dose of a therapeutic agonistic antibody that specifically binds an immunostimulatory receptor or a schedule of antibody administration for treating a subject having cancer, the method comprising

(a) Administering the agonistic antibody to an animal model (e.g., a mouse tumor model, a monkey tumor model);

(b) obtaining a sample (e.g., blood) from the animal model;

(c) determining RO for the antibody in the sample;

(d) using the RO obtained from step (c) to predict/predict an expected RO in the subject; and

(e) an amount of antibody or schedule of antibody administration sufficient to achieve and/or maintain a RO or range of RO of less than about 80% (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, less than about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30%) is selected.

In certain embodiments, an effective amount of an antibody is defined based on the range of ROs at which the antibody exhibits therapeutic efficacy (e.g., anti-tumor activity). Accordingly, the present application also provides a method for determining the effective RO range of an agonistic antibody that specifically binds to an immunostimulatory receptor, which antibody achieves a therapeutic effect, e.g., anti-tumor activity, within this range, comprising

(a) The antibodies are administered in varying amounts or at varying frequencies, for example, to an animal model (e.g., a mouse tumor model).

(b) A sample (e.g., blood) is obtained from the animal.

(c) Measuring RO of the antibody in the sample to obtain a dose-response RO curve.

(d) The amount or frequency of administration of antibody that produces a therapeutic effect (e.g., anti-tumor activity) is determined and correlated to a range of ROs.

In some embodiments, the method further comprises the step of predicting/predicting the expected range of RO for the agonistic antibody for the subject (e.g., human patient) based on the range of RO determined in step (d).

In some embodiments, the present application provides a method of selecting an effective dose of a therapeutic agonistic antibody that specifically binds an immunostimulatory receptor or a schedule of antibody administration for treating a subject having cancer, the method comprising

(a) A sample (e.g., a tumor biopsy) is obtained from a patient treated with the antibody.

(b) Determining RO of said antibody in said sample.

(c) Using the RO obtained from step (b) to determine an expected RO in the subject; and

(d) an amount of antibody or schedule of antibody administration sufficient to achieve and/or maintain less than about 80% (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, less than about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30%) is selected.

In some embodiments, the expected RO and/or RO range in a human patient can be predicted/predicted from RO and/or RO range associated with therapeutic efficacy (e.g., anti-tumor activity) from preclinical data, or by retrospective analysis.

In a preclinical setting, one can, for example, use dissociation constants Kd determined from surface plasmon resonance or bound EC obtained from in vitro human cell lines₅₀Human RO was calculated (Muller PY and Brennan FR, Clin Pharmacol Ther.2009; 85: 247-58; Saber H et al, Regul Toxicol Pharmacol.2016; 81:448-56). however, due to the limitations of various in vitro systems (e.g., lack of competing ligands), prediction of human RO can be supported by assessing how relevant in vitro RO in an animal species correlates with in vitro RO observed in vivo in that species (Yang Z et al, JPharmacol Exp Ther.2015; 355:506 Across 515). Clinically, population pharmacokinetic-pharmacodynamic (PK-PD) modeling can be performed to determine the relationship between drug concentration and RO data (Rosario MC et al, Br J Clin Pharmacol.2008; 65:86-94), from which human RO data can be predicted at different dosage regimens. An effective human dose of agonistic therapeutic antibody or schedule of antibody administration may then be determined using the predicted/predicted RO, e.g., a dose or frequency sufficient to achieve and/or maintain a range of RO or RO in a human cancer patient of less than about 80% (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30%).

Suitable preclinical animal models for predicting expected RO and/or range of RO for agonistic antibodies that bind to an immunostimulatory receptor include, but are not limited to, mouse tumor models (e.g., CT26 mouse syngeneic tumor model), mouse vaccination models, monkey vaccination models, and in vitro stimulation model systems using a native human leukocyte population.

For retrospective analysis, RO can be determined in a sample of a patient treated with an agonistic antibody that binds to an immunostimulatory receptor. Samples from patients may be, for example, tumor biopsies, blood and isolated peripheral blood mononuclear cells. The RO obtained by retrospective analysis can then be used to provide information on the dose or schedule of administration to the human cancer patient (e.g., a dose or frequency sufficient to achieve and/or maintain less than about 80% of the RO).

In some embodiments, the present application provides methods of treating cancer comprising administering to a subject in need thereof an effective amount of a therapeutic agonistic antibody that specifically binds to an immunostimulatory receptor or a schedule of antibody administration, wherein the amount of antibody to be administered or the schedule of antibody administration is selected according to the above-described dose selection methods.

In certain embodiments, the present application provides methods of treating cancer in a subject (e.g., a human patient), wherein an amount of a therapeutic agonistic antibody or schedule of antibody administration sufficient to achieve and/or maintain a range of RO and/or RO of less than about 80% (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, less than about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%) has been determined for the subject using the methods described herein, comprising administering said sufficient amount of said therapeutic agonistic antibody or schedule of administration to the subject.

The present application also provides a method of monitoring the level of a therapeutic agonistic antibody that specifically binds to an immunostimulatory receptor in a subject undergoing treatment for cancer, comprising:

(a) obtaining a sample (e.g., blood) from a subject;

(b) determining RO (i.e. the occupancy of the receptor by the antibody) in the sample;

(c) decreasing the amount or frequency of administration of the antibody to the subject if the receptor occupancy is greater than about 80%, or increasing the amount or frequency of the antibody if the receptor occupancy is less than about 20%;

(d) optionally repeating steps (a) - (c) until a sufficient dose of antibody or schedule of antibody administration is reached to achieve and/or maintain about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30% RO.

In another embodiment, the present application provides a method of monitoring the level of a therapeutic agonistic antibody that specifically binds to an immunostimulatory receptor in a subject undergoing treatment for cancer, comprising:

(a) obtaining a sample (e.g., blood) from a subject;

(b) determining RO (i.e. the occupancy of the receptor by the antibody) in the sample;

(c) establishing a PK-PD relationship between antibody concentration and RO for predicting RO for dose regimen and/or dose frequency;

(d) the dosage and/or schedule of antibody administration is selected to achieve and/or maintain an RO and/or range of RO of about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30%.

The present application also provides methods of treating cancer comprising administering to a subject in need thereof an agonistic antibody that specifically binds to an immunostimulatory receptor and an adjunct therapy. Wherein the additional therapy is administered at a fixed frequency and the agonistic antibody is administered at a frequency sufficient to achieve and/or maintain a RO and/or range of RO of less than about 80% (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, less than about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30%). In some embodiments, the frequency of administration of agonistic antibodies is determined using the dose selection methods described above.

In some embodiments of the methods described herein, the immunostimulatory receptor is a co-stimulatory receptor, for example a receptor selected from the group consisting of: members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), ICOS (CD278), CD28, LIGHT, CD40L, TIM1, SLAM, CD1, CD2, CD226, LFA-1(CD11A, CD18), CD2, CD5, CD7, CD30, CD54, CD97, CD154, CD160, LIGHT, NKG2C, SLAMF7 and NKp 80.

In some embodiments, the co-stimulatory receptor is a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF). Accordingly, in some embodiments, the agonistic antibody used in the methods described herein is conjugated to TNFR1, TNFR2, HVEM, LT β R, OX40, CD27, CD40, FAS, DCR3, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, OPG, RANK, FN14, TACI, BAFFR, BCMA, GITR, TROY, DR3 (death receptor 3), DR6 (death receptor 6), xedr (ectodisplsin) a2 receptor), or NGFR.

In a particular embodiment, the immunostimulatory receptor is OX40. Accordingly, the present application provides a method of treating cancer, comprising administering to a subject in need thereof an agonist antibody that specifically binds OX40 (e.g., OX40.21), wherein the agonist antibody is administered in an amount or frequency sufficient to achieve and/or maintain less than about 80% (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, less than about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30%) of RO and/or a range of RO in the subject.

In some embodiments, the methods of treating cancer described herein comprise administering to a subject in need thereof an effective amount of an anti-OX 40 antibody and an effective amount of an anti-PD-1 antibody, wherein the anti-OX 40 and the anti-PD-1 antibody are administered for at least one administration cycle, wherein the cycle is 12 weeks, wherein for each cycle of the at least one cycle, at least one dose of the anti-OX 40 antibody is administered at a fixed dose of about 1,3, 10, 20, 40, 50, 80, 100, 130, 150, 180, 200, 240, or 280mg and at least 3 doses of the anti-PD-1 antibody is administered at a fixed dose of about 50, 80, 100, 120, 150, 180, 200, 240, 480, 720, or 960 mg. In one embodiment, for each cycle of the at least one cycle, one dose of the anti-OX 40 antibody is administered at a fixed dose of about 20, 40, or 80mg and 3 doses of the anti-PD-1 antibody are administered at a fixed dose of about 480 mg. In one embodiment, for each cycle of the at least one cycle, one dose of the anti-OX 40 antibody is administered at a fixed dose of about 20mg and 3 doses of the anti-PD-1 antibody are administered at a fixed dose of about 480 mg. In one embodiment, for each cycle of the at least one cycle, one dose of the anti-OX 40 antibody is administered at a fixed dose of about 40mg and 3 doses of the anti-OX 40 antibody are administered at a fixed dose of about 480. In one embodiment, for each cycle of the at least one cycle, one dose of the anti-OX 40 antibody is administered at a fixed dose of about 80mg and 3 doses of the anti-PD-1 antibody are administered at a fixed dose of about 480. In one embodiment, the anti-PD-1 antibody is administered on days 1, 29, and 57 of each cycle. In one embodiment, the anti-OX 40 antibody is administered on day 1 of each cycle. In one embodiment, the anti-PD-1 antibody is administered on days 1, 29, and 57 of each cycle and the anti-OX 40 antibody is administered on day 1 of each cycle. In one embodiment, the 12 week administration cycle may be repeated as desired. In one embodiment, administration consists of up to 9 cycles. In one embodiment, administration comprises 1, 2,3, 4,5, 6, 7, 8, or 9 cycles. In one embodiment, the OX-40 antibody comprises OX 40.21. In one embodiment, the anti-PD-1 antibody comprises nivolumab. In one embodiment, the cancer is selected from the group consisting of bladder cancer, cervical cancer, renal cell carcinoma, testicular cancer, colorectal cancer, lung cancer, head and neck cancer, and ovarian cancer. In one embodiment, the cancer is bladder cancer. In one embodiment, the subject is a human subject.

Combination therapy of an agonistic antibody that binds an immunostimulatory receptor with an additional agent is also provided. In such embodiments, the effective amount of an agonist antibody (e.g., an anti-OX 40 antibody) may be substantially less than if the agonist antibody was used alone (i.e., at the time of monotherapy).

Accordingly, the present application provides a method of treating cancer comprising administering to a subject in need thereof an agonist antibody (e.g., MEDI6469, MEDI0562, PF-04518600, MOXR0916, GSK3174998, and the antibody described in WO2016/196228 (e.g., OX40.21)) that specifically binds to OX40 and an additional therapy (non-limiting examples include anti-PD 1 antibodies, anti-PDL 1 antibodies, anti-LAG 3 antibodies, anti-CTLA 4 antibodies, and anti-TGF β antibodies), wherein the agonist antibody is administered in an amount or frequency sufficient to achieve and/or maintain a range of RO and/or RO of less than about 80% (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, less than about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30%).

In some embodiments, the anti-OX 40 antibody is administered at a different frequency than the adjunctive therapy. For example, the additional therapy is administered at a fixed frequency, while the anti-OX 40 antibody is administered in an amount or frequency sufficient to achieve less than about 80% (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, less than about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30%) of RO and/or RO range.

In some embodiments, an agonist antibody that binds to an immunostimulatory receptor (e.g., an anti-OX 40 antibody, such as OX40.21) is pulsed when used in combination with an additional therapy (e.g., nivolumab). In some embodiments, the pulsed administration of the agonistic antibody is to achieve and/or maintain less than about 80% (or less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 50%, less than about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%) RO. For example, in one embodiment, pulsed administration may involve a combination therapy of an agonistic antibody with an additional agent, wherein the agonistic antibody is administered once every 8 or 12 weeks and the additional agent (e.g., anti-PD 1 antibody) is administered once every 4 weeks.

Markers of T cell activation can be monitored in a subject treated with an agonistic antibody that binds an immunostimulatory receptor to confirm that the agonistic antibody is being administered at an effective dose or frequency. Examples of other markers of T cell activation that can be monitored (and that exhibit a "hook effect") include, but are not limited to, surface expression of immunostimulatory receptors (e.g., OX40), ICOS, CD44, and Ki67, as well as cytokines (e.g., IFN- γ, IL-2) known to be upregulated during immune responses. Methods for measuring the levels of the above markers are well known in the art (e.g., ELISA). T cell proliferation can be monitored by, for example, a 3[ H ] -thymidine binding assay.

When the immunostimulatory receptor targeted in the methods described herein is OX40, soluble OX40(sOX40) can be used as a marker to monitor the efficacy of agonist antibody therapy because sOX40 exhibits the so-called "hook effect" at high RO levels (see, e.g., example 8). An exemplary method (ELISA) for determining the level of sOX40 is provided in example 8.

Cancers whose growth can be treated or monitored by the methods described herein include cancers that are generally responsive to immunotherapy, as well as cancers that are generally non-responsive to immunotherapy. The cancer may be a cancer with a solid tumor or a hematologic malignancy (liquid tumor). Non-limiting examples of cancers for treatment include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, hepatocellular carcinoma, colorectal cancer, endometrial cancer, renal cancer (e.g., Renal Cell Carcinoma (RCC), prostate cancer (e.g., hormone refractory prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, and head and neck cancer (or carcinoma), gastric cancer, germ cell tumor, pediatric sarcoma, paranasal sinus natural killer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, and the like, Uterine cancer, cancer of the anal region, cancer of the testis, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, carcinoma of the esophagus, carcinoma of the small intestine, carcinoma of the endocrine system, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, sarcoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, solid tumors of childhood, carcinoma of the ureter, carcinoma of the renal pelvis, tumors of the Central Nervous System (CNS), primary central nervous system lymphomas, tumor angiogenesis, rachiomas, brain cancers, brain stem gliomas, pituitary adenomas, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including asbestos-induced), virus-related cancers or virally-derived cancers (such as human papilloma virus (HPV-related or HPV-derived tumors)), and cancers derived from two major blood cell lineages, namely the myeloid lineage (granulocytes, Platelet cells, macrophages and mast cells) or a lymphoid cell line (producing B cells, T cells, NK cells and plasma cells), such as all types of leukemia, lymphoma and myeloma, for example: acute, chronic, lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL) and Chronic Myelogenous Leukemia (CML), undifferentiated AML (M0), myelogenous leukemia (M1), myelogenous leukemia (M2; cell maturation), promyelocytic leukemia (M3 or M3 variant [ M3V ]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [ M4E ]), monocytic leukemia (M5), erythrocytic leukemia (M6), cytomegaloleukemia (M7), solitary myelosarcoma and chloroma; lymphomas, such as Hodgkin Lymphoma (HL), non-hodgkin lymphoma (NHL), B-cell hematologic malignancies, such as B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytic B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphocytes; and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, genuine histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, burkitt's lymphoma, follicular lymphoma, Diffuse Histiocytic Lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also known as mycosis fungoides or Sezary syndrome), and lymphoplasmacytic lymphoma (LPL) with Waldenstrom macroglobulinemia. Myelomas, such as IgG-type myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also known as indolent myeloma), orphan plasmacytoma, and multiple myeloma, Chronic Lymphocytic Leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma, seminoma, teratoma, tumors of the central and peripheral nerves, including astrocytoma, schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer, and teratoma; hematopoietic tumors of lymphocyte lineage, such as T cell and B cell tumors, including but not limited to T cell disorders such as T-prolymphocytic leukemia (T-PLL), including but not limited to small cell type and gyrocellular type; large granular cell lymphocytic leukemia (LGL), preferably of the T cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (polymorphic and immunoblastic subtypes); angiocentric (nasal) T cell lymphoma; head and neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of said cancers.

The methods described herein can also be used to treat metastatic cancer, unresectable and/or refractory cancer (e.g., cancer refractory to past immunotherapy, e.g., treatment with a blocking CTLA-4 or PD-1 antibody), and recurrent cancer.

In some embodiments, a patient to be treated with a method disclosed herein has an advanced solid tumor. For example, in one embodiment, the patient has cervical cancer. In another embodiment, the patient has colorectal cancer (CRC). In another embodiment, the patient has bladder cancer (e.g., unresectable locally advanced or metastatic bladder cancer). In another embodiment, the patient has ovarian cancer (e.g., unresectable locally advanced or metastatic ovarian cancer).

In one embodiment, the patient treated with the methods described herein has non-small cell lung cancer (NSCLC). In another embodiment, the patient has squamous cell carcinoma of the head and neck (SCCHN). In another embodiment, the patient has a B-cell non-Hodgkin's lymphoma (B-NHL). In another embodiment, the patient has myeloma. In another embodiment, the patient has melanoma. In another embodiment, the patient has diffuse large B-cell lymphoma (DLBCL).

In one embodiment, the patient treated with the methods described herein has cervical cancer. In one embodiment, the cervical cancer is unresectable, metastatic, or recurrent cervical cancer with documented disease progression.

In one embodiment, the patient treated with the methods described herein has renal cell carcinoma. In one embodiment, the renal cell carcinoma is metastatic renal cell carcinoma. In one embodiment, the renal cell carcinoma is a renal cell carcinoma having a clear cell component.

In one embodiment, the patient treated with the methods described herein has testicular cancer.

In one embodiment, the patient being treated with the methods described herein has colorectal cancer. In one embodiment, the colorectal cancer is microsatellite instability high (MSI-H) colorectal cancer. In one embodiment, the colorectal cancer is microsatellite-stabilized colorectal cancer. In one embodiment, the colorectal cancer is a mismatch repair deficient colorectal cancer.

In one embodiment, the patient treated with the methods described herein has lung cancer.

In one embodiment, the patient treated with the methods described herein has a head and neck cancer. In one embodiment, the head and neck cancer is squamous cell carcinoma.

In one embodiment, the patient treated with the methods described herein has ovarian cancer. In one embodiment, the ovarian cancer is unresectable locally advanced ovarian cancer. In one embodiment, the ovarian cancer is metastatic ovarian cancer. In one embodiment, the ovarian cancer is recurrent platinum-sensitive ovarian cancer.

In some embodiments, a patient treated with a method described herein is a patient with a cancer that exhibits an inadequate response to a previous treatment (e.g., a previous immunooncology drug treatment), or a cancer that is refractory or resistant, wherein the refractory or resistant state of the cancer is intrinsic, or the refractory or resistant state is acquired a posteriori. In some embodiments, the patient has not previously received (i.e., has not used) an immunooncology drug, e.g., a PD-1 pathway antagonist.

In some embodiments, the methods described herein can further include standard of care treatments (e.g., surgery, radiation therapy, and chemotherapy). In other embodiments, the methods can be as a maintenance therapy, e.g., a therapy intended for preventing the occurrence or recurrence of a tumor.

In some embodiments, an agonist antibody that binds an immunostimulatory receptor such as TNF and a TNFR family molecule (e.g., OX40) is administered to a subject as an adjunct therapy. In some embodiments, agonist antibodies that bind to immunostimulatory receptors such as TNF and TNFR family molecules (e.g., OX40) are used as first, second or third line therapies.

In some embodiments, an agonist antibody that binds an immunostimulatory receptor, such as TNF and TNFR family molecules (e.g., OX40), can be administered as a monotherapy or as the sole immunostimulatory therapy.

In other embodiments, agonist antibodies that bind to immunostimulatory receptors such as TNF and TNFR family molecules (e.g., OX40) can be administered as part of a combination therapy, as described below.

Agonist antibodies that specifically bind to immunostimulatory receptors, such as TNF and TNFR family molecules (e.g., OX40), can be combined with immunooncology agents, such as (i) agonists of immunostimulatory (e.g., co-stimulatory) molecules (e.g., receptors or ligands), and/or (ii) antagonists of immunosuppressive molecules (e.g., receptors or ligands), on immune cells (e.g., T cells), all of which can result in an amplified immune response, e.g., an antigen-specific T cell response. In certain aspects, the immunooncology agent is (i) an agonist of an immunostimulatory (including co-stimulatory) molecule (e.g., a receptor or ligand), or (ii) an antagonist of an immunosuppressive (including co-inhibitory) molecule (e.g., a receptor or ligand), present on a cell involved in innate immunity, such as an NK cell. Such immune oncology agents are often referred to as immune checkpoint modulators, e.g., immune checkpoint inhibitors or immune checkpoint stimulants.

In some embodiments, an agonist antibody that specifically binds to an immunostimulatory receptor such as TNF and TNFR family molecules (e.g., OX40) is administered with an agent that targets a stimulatory or inhibitory molecule belonging to an immunoglobulin superfamily member (IgSF). In some embodiments, an agonistic antibody that specifically binds to an immunostimulatory receptor may be administered with an agent that targets (or specifically binds to) a member of the B7 membrane-bound ligand family, which includes B7-1, B7-2, B7-H1(PD-L1), B7-DC (PD-L2), B7-H2(ICOS-L), B7-H3, B7-H4, B7-H5(VISTA), and B7-H6, or a costimulatory or co-inhibitory receptor that specifically binds to a member of the B7 family.

In some embodiments, an agent that specifically binds to an immunostimulatory receptor such as TNF and TNFR family molecules (e.g., OX40) may also be administered with an agent that targets a member of the TNF and TNFR family molecules (ligands or receptors), such as CD40 and CD40L, GITR-L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, kl, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, xedr, TACI, APRIL, BCMA, hvlt β R, LIGHT, DcR3, em, VEGI/TL1 DR 585, TRAMP/DR3, FAS 1, TNFR1, TNFR β 1, TNF β R1, TNF α toxin, TNF α β R599, TNF α toxin, TNF α β 9, TNF α toxin, faq β 9, faq β 9, faq β, faq, fag.

In some embodiments, an agonist antibody that specifically binds an immunostimulatory receptor such as TNF and TNFR family molecules (e.g., OX40) is administered with one or more of the following agents:

(1) antagonists (inhibitors or blockers) of proteins that inhibit T cell activation (e.g. immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L1, PD-L2 and LAG-3, as described above, and any of the following proteins: TIM-3, galectin 9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1 and TIM-4; and/or

(2) Agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS-L, GITR-L, CD70, CD27, CD40, DR3, and CD 28H.

Exemplary agents for treating cancer that modulate one of the above proteins include Yervoy^TM(ipilimumab or Tremelimumab (anti-CTLA-4), galiximab (galiximab) (anti-B7.1), BMS-936558 (anti-PD-1), MK-3475 (anti-PD-1), AMP224 (anti-B7 DC), BMS-936559 (anti-B7-H1), MPDL3280A (anti-B7-H1), MEDI-570 (anti-ICOS), AMG557 (anti-B7H 2), MGA271 (anti-B7H 3), IMB21 (anti-LAG-3), BMS-663513 (anti-CD 137), PF-05082566 (anti-CD 137), CDX-1127 (anti-CD 27), Atacept (anti-TACI), CP-P870893 (anti-CD 40), Lucateumumab (Lucateumumab) (anti-CD 40), Damotuzumab (anti-CTLA 369634), anti-MUTUMAb (anti-CD 3), Mutuzumab (anti-MUTUMORE). In some embodiments, an agonistic antibody that specifically binds an immunostimulatory receptor is administered with pidilizumab (CT-011).

Agonist antibodies that specifically bind to immunostimulatory receptors such as TNF and TNFR family molecules (e.g., OX40) may be used in combination with other molecules for cancer treatment including antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells, e.g., antagonists of KIRs (e.g., lirilumab).

In certain embodiments, an agonist antibody that specifically binds an immunostimulatory receptor such as TNF and TNFR family molecules (e.g., OX40) can be administered with an antagonist of a cytokine that inhibits T cell activation or an agonist of a cytokine that stimulates T cell activation.

In certain embodiments, agonist antibodies that specifically bind to immunostimulatory receptors such as TNF and TNFR family molecules (e.g., OX40) can be used in combination with the following (i) and/or (ii) to stimulate an immune response, e.g., for treating proliferative diseases such as cancer: (i) an antagonist (or inhibitor or blocker) of a "protein of the IgSF family, or B7 family, or TNF family that inhibits T cell activation"; or "cytokines that inhibit T cell activation (e.g., IL-6, IL-10, TGF- β, VEGF;" immunosuppressive cytokines "); and/or (ii) an agonist of a "stimulatory receptor of the IgSF family, B7 family or TNF family", or of a "receptor of a cytokine that stimulates T cell activation".

Other agents suitable for combination therapy include agents that inhibit or deplete macrophages or monocytes including, but not limited to, CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716, WO 13/132044); or FPA-008(WO 11/140249; WO 13169264; WO 14/036357).

Agonist antibodies that specifically bind to immunostimulatory receptors such as TNF and TNFR family molecules (e.g., OX40) may also be administered with agents that inhibit TGF- β signaling.

Other agents for combination therapy include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, GM-CSF-secreting cell vaccines, CpG oligonucleotides and imiquimod, or therapies that enhance tumor cell immunogenicity (e.g., anthracyclines).

Other therapies that may be used in combination therapy include therapies that deplete or block Treg cells, for example, agents that specifically bind CD 25.

Another class of therapies that can be used in combination with agonistic antibodies that specifically bind to immunostimulatory receptors such as TNF and TNFR family molecules (e.g., OX40) are therapies that inhibit metabolic enzymes such as Indoleamine Dioxinase (IDO), dioxinase, arginase, or nitric oxide synthase.

Another class of drugs that can be used with agonistic antibodies that specifically bind to immunostimulatory receptors such as TNF and TNFR family molecules (e.g., OX40) include drugs that inhibit the formation of adenosine or inhibit the adenosine A2A receptor.

Other therapies for combination therapy include therapies to reverse/prevent T cell anergy or failure, and therapies that elicit innate immune activation and/or inflammation at the tumor site.

In some embodiments, agonist antibodies that specifically bind to immunostimulatory receptors such as TNF and TNFR family molecules (e.g., OX40) can be combined with more than one immunooncology agent, and can be combined with, for example, a combinatorial strategy that targets multiple elements of the immune pathway, e.g., one or more of the following: therapies that enhance tumor antigen presentation (e.g., dendritic cell vaccines, GM-CSF secreting cell vaccines, CpG oligonucleotides, imiquimod); therapies that inhibit negative immune regulation, for example by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathways and/or depleting or blocking Tregs or other immunosuppressive cells; therapies that stimulate positive immunomodulation, e.g., agonists that stimulate the CD-137 and/or GITR pathway and/or stimulate T cell effector function; therapies that systematically increase the frequency of anti-tumor T cells; a therapy to deplete or inhibit Tregs, e.g., in tumors, e.g., using an antagonist of CD25 (such as daclizumab) or by ex vivo anti-CD 25 bead depletion; therapies that affect inhibitory myeloid cell function in tumors; therapies that increase the immunogenicity of tumor cells (e.g., anthracycline drugs); adoptive T cell or NK cell transplantation, including genetically modified cells, such as cells modified by chimeric antigen receptors (CAR-T therapy); therapies that inhibit metabolic enzymes such as indoleamine-dioxin enzyme (IDO), dioxin enzyme, arginase, or nitric oxide synthase; therapies to reverse/prevent T cell anergy or failure; triggering therapy of innate immune activation and/or inflammation at a tumor site; administering an immunostimulatory cytokine; or block immunosuppressive cytokines.

In some embodiments, agonist antibodies that specifically bind to immunostimulatory receptors such as TNF and TNFR family molecules (e.g., OX40) can be used with one or more of the following: agonists linked to positive co-stimulatory receptors, blockers that attenuate signaling through inhibitory receptors, antagonists and one or more agents that systematically increase the frequency of anti-tumor T cells, agents that overcome unique immunosuppressive pathways in the tumor microenvironment (e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1 interaction), deplete or inhibit Tregs (e.g., depletion using anti-CD 25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD 25 beads), inhibit metabolic enzymes such as IDO, or reverse/prevent the inability or failure of T cells); and agents that trigger innate immune activation and/or inflammation at the tumor site.

In certain embodiments, if the subject is BRAF V600 mutation positive, an agonistic antibody that specifically binds to an immunostimulatory receptor such as TNF and TNFR family molecules (e.g., OX40) is administered with a BRAF inhibitor.

In certain embodiments, an agonistic antibody that specifically binds an immunostimulatory receptor such as TNF and a TNFR family molecule (e.g., OX40) is administered with another immunostimulatory antibody, e.g., an antagonistic anti-PD 1 antibody, an antagonistic anti-PDL 1 antibody, an antagonistic anti-CTLA 4 antibody, an antagonistic anti-LAG 3 antibody; the anti-OX 40 antibody is administered with another immunostimulatory antibody. In a particular embodiment, the combination therapy comprises an agonist anti-OX 40 antibody and an antagonist anti-PD 1 antibody.

Suitable PD-1 antagonists for use in the methods described herein include, but are not limited to, ligands, antibodies (e.g., monoclonal antibodies and bispecific antibodies), and multivalent agents. In one embodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusion protein, e.g., AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody. An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibody comprising the CDRs or variable regions of one of antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4 and 4a11 described in WO 2006/121168. In certain embodiments, the anti-PD 1 antibody is MK-3475(Lambrolizumab) described in WO 2012/145493; and AMP-514 as described in WO 2012/145493. Other known PD-1 antibodies and other PD-1 inhibitors include antibodies described in WO 2009/014708, WO 03/099196, WO2009/114335, WO2011/066389, WO 2011/161699, WO2012/145493, U.S. patent nos. 7,635,757 and 8,217,149, and U.S. patent publication No. 2009/0317368. Any of the anti-PD-1 antibodies disclosed in WO2013/173223 may also be used. Antibodies that compete with one of these antibodies for binding to PD-1 or/and bind to the same epitope on PD-1 may also be used in combination therapy. Another approach to targeting the PD-1 receptor is a recombinant protein, termed AMP-224, fused to the Fc portion of IgG1 from the extracellular domain of PD-L2 (B7-DC). In certain embodiments, the antibody has at least about 90% amino acid sequence identity to a variable region of the antibody described above.

In certain embodiments, an agonistic antibody that specifically binds an immunostimulatory receptor such as TNF and TNFR family molecules (e.g., OX40) is used in combination with nivolumab, which comprises heavy and light chains comprising the sequences set forth in SEQ ID NOs 16 and 17, respectively, or an antigen-binding fragment and variants thereof. In certain embodiments, the antibody has the heavy and light chain CDRs or variable regions of nivolumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH of nivolumab having the sequence set forth in SEQ ID NO. 18 and the CDR1, CDR2 and CDR3 domains of the VL of nivolumab having the sequence set forth in SEQ ID NO. 19. In certain embodiments, the antibody comprises: CDR1, CDR2 and CDR3 domains comprising the sequences shown in SEQ ID NO. 20-22, respectively, and CDR1, CDR2 and CDR3 domains comprising the sequences shown in SEQ ID NO. 23-25, respectively. In certain embodiments, the antibody comprises VH and/or VL regions comprising the amino acid sequences set forth in SEQ ID NO:18 and/or SEQ ID NO:19, respectively. In certain embodiments, the antibody has at least about 90%, e.g., at least about 90%, 95%, or 99% variable region identity to SEQ ID No. 18 and/or SEQ ID No. 19.

Exemplary anti-PD-L1 antibodies include BMS-936559 (referred to as 12a4 in WO 2007/005874 and U.S. patent No.7,943,743), or antibodies that include the CDRs or variable regions of 3G10, 12a4, 10a5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, which are described in PCT publication WO 07/005874 and U.S. patent No.7,943,743. In certain embodiments, the anti-PD-L1 antibody is MEDI4736 (also known as anti-B7-H1), MPDL3280A (also known as RG7446), MSB0010718C (WO2013/79174), or rHigM12B 7. Any of the anti-PD-L1 antibodies disclosed in WO2013/173223, WO2011/066389, WO2012/145493, U.S. patent nos. 7,635,757 and 8,217,149, and U.S. publication No. 2009/145493 may also be used.

Exemplary anti-CTLA-4 antibodies include Yervoy^TM(ipilimumab or antibody 10D1, described in PCT publication WO 01/14424), tremelimumab (formerly ticilimumab, CP-675, 206), or anti-CTLA-4 antibodies optionally described in the following publications: WO 98/42752; WO 00/37504; U.S. patent No. 6,207,156; hurwitz et al (1998) Proc. Natl. Acad. Sci. USA 95(17) 10067-; camacho et al (2004) J.Clin.Oncology 22(145) Abstract No.2505 (antibody CP-675206); and Mokyr et al (1998) Cancer Res.58: 5301-. Any of the anti-CTLA-4 antibodies disclosed in WO2013/173223 may also be used.

In certain embodiments, an agonistic antibody that specifically binds to an immunostimulatory receptor is used in combination with ipilimumab. In certain embodiments, the antibody has the heavy and light chain CDRs or variable regions of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of the VH of ipilimumab having the sequence set forth in SEQ ID NO. 26 and the CDR1, CDR2 and CDR3 domains of the VL of ipilimumab having the sequence set forth in SEQ ID NO. 27. In certain embodiments, the antibody comprises: CDR1, CDR2 and CDR3 domains comprising the sequences set forth in SEQ ID NOS.28-30, respectively, and CDR1, CDR2 and CDR3 domains comprising the sequences set forth in SEQ ID NOS.31-33, respectively. In certain embodiments, the antibody comprises a VH and/or VL region comprising the amino acid sequences set forth in SEQ ID NO 26 and/or SEQ ID NO 27, respectively. In certain embodiments, the antibody has at least about 90%, e.g., at least about 90%, 95%, or 99% variable region identity to SEQ ID No. 26 or SEQ ID No. 27.

Exemplary anti-LAG 3 antibodies include antibodies comprising the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5, which are described in U.S. patent publications nos. US2011/0150892, WO10/19570, and WO 2014/008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other anti-LAG-3 antibodies known in the art may be used including IMP731 and IMP-321, which are described in US2011/007023, WO08/132601 and WO 09/44273.

In certain embodiments, the combination of therapeutic antibodies discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions of each antibody in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies may be administered continuously. Furthermore, if more than one dose of the combination therapeutic antibody is administered sequentially, the order of sequential administration may be reversed or remain the same at each point in time of administration, and sequential administration may be combined with simultaneous administration, or any combination thereof.

In certain embodiments, a subject having a disease that may benefit from stimulation of the immune system, such as cancer or an infectious disease, is treated by administering to the subject an agonistic antibody that specifically binds to an immunostimulatory receptor and an immunooncology agent. Exemplary immunooncology agents include CD137(4-1BB) agonists (e.g., agonistic CD137 antibodies, such as urelumab or PF-05082566(WO 12/32433))); GITR agonists (e.g., agonistic anti-GITR antibodies), CD40 agonists (e.g., agonistic CD40 antibodies); CD40 antagonists (e.g., antagonistic CD40 antibodies, such as urelumab or PF-05082566(WO 12/32433)); GITR agonist (e.g., an agonistic anti-GITR antibody), CD40 agonist (e.g., an agonistic CD40 antibody); a CD40 antagonist (e.g., an antagonistic CD40 antibody such as lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870,893, or Chi Lob 7/4); CD27 agonists (e.g., agonistic CD27 antibodies such as varliumab (CDX-1127)), MGA271 (anti-B7H 3) (WO 11/109400)); KIR antagonists (e.g., lirilumab); IDO antagonists (e.g., INCB-024360(WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoimod (indoximod), NLG-919(WO09/73620, WO09/1156652, WO11/56652, WO12/142237) or F001287); toll-like receptor agonists (e.g., TLR2/4 agonists (e.g., BCG); TLR7 agonists (e.g., Hiltonol or imiquimod); TLR7/8 agonists (e.g., resiquimod); or TLR9 agonists (e.g., CpG 7909)); and TGF- β inhibitors (e.g., GC1008, LY2157299, TEW7197, or IMC-TR 1).

Optionally, an agonistic antibody that specifically binds an immunostimulatory receptor, as the sole immunotherapeutic agent, or as a combination of the agonistic antibody with one or more additional immunotherapeutic antibodies (e.g., anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 blocking agent), may be further combined with an immunogenic agent, such as a cancerous cell, a purified tumor antigen (including recombinant proteins, peptides, and sugar molecules), a cell, and a cell transfected with a gene encoding an immunostimulatory cytokine (He et al, (2004) j.immun.173: 4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100, MAGE antigens, Trp-2, MART1, and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below). The combination of an agonistic antibody that specifically binds to an immunostimulatory receptor and one or more additional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade) may also be further combined with standard cancer treatment methods. For example, a combination of an agonistic antibody that specifically binds an immunostimulatory receptor and one or more additional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade) may be effectively combined with a chemotherapeutic regimen. In these cases, the dose of the other chemotherapeutic agent administered with the combination may be reduced (Mokyr et al (1998) Cancer Research 58: 5301-5304). For example, such associations may include: agonistic antibodies that specifically bind to immunostimulatory receptors, with or without additional antibodies (e.g., anti-CTLA-4 antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodies and/or anti-LAG-3 antibodies), further in combination with dacarbazine or interleukin-2 (IL-2), for the treatment of melanoma. The scientific rationale for blocking binding of CTLA-4 and/or PD-1 and/or PD-L1 and/or PD-L1 and/or LAG-3 to chemotherapy with agonistic antibodies that specifically bind to immunostimulatory receptors is that cell death, which is a result of the cytotoxic effects of most chemotherapeutic compounds, should result in increased levels of tumor antigens in the antigen presentation pathway. Other combination therapies which may produce a synergistic effect by cell death in combination with agonistic antibodies which specifically bind to immunostimulatory receptors (with or without blockade by CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3) include radiation, surgery or hormone deprivation. Both of these procedures produce a source of tumor antigens in the host. The angiogenesis inhibitor may also be combined with an agonistic antibody that specifically binds to an immunostimulatory receptor in combination with a CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade. Inhibition of angiogenesis results in tumor cell death, which may be the source of tumor antigens that feed into the host antigen presentation pathway.

In certain embodiments, agonistic antibodies that specifically bind to immunostimulatory receptors may be used as the sole immunotherapeutic agent, or in combination with anti-OX 40 antibodies and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocking antibodies, may be used in combination with bispecific antibodies (U.S. patent nos. 5,922,845 and 5,837,243) that target Fc α or Fc γ receptor-expressing effector cells to tumor cells. Bispecific antibodies can be used to target two different antigens. The T cell arm of these responses will be enhanced by the use of agonistic antibodies that specifically bind to the immunostimulatory receptor in combination with CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade.

In another embodiment, the agonistic antibody that specifically binds to an immunostimulatory receptor may serve as the sole immunotherapeutic agent, or the agonistic antibody that specifically binds to an immunostimulatory receptor may be combined with an additional immunostimulatory agent, e.g., an antagonistic anti-CTLA-4 antibody and/or an antagonistic PD-1 antibody and/or an antagonistic PD-L1 antibody and/or an antagonistic LAG-3 agent (e.g., an antibody), which may be used in combination with an anti-tumor antibody, e.g., Rituxan (Rituxan)

(rituximab), Herceptin (Herceptin)

(trastuzumab), Bexxar

(tositumomab), Zevalin

(ibritumomab tiuxetan), Campath

(alemtuzumab), Lymphocide

(epratuzumab), Avastin

(bevacizumab) and Tarceva (Tarceva)

(erlotinib), and the like. By way of example, and without wishing to be bound by theory, treatment with an anti-cancer antibody or a conjugate of an anti-cancer antibody and a toxin may lead to cancer cell (e.g., tumor cell) death, which will enhance the immune response mediated by an immunostimulant (e.g., an antagonistic CTLA-4, PD-1, PD-L1, or LAG-3 agent, e.g., an antibody). In an exemplary embodiment, treatment of a proliferative disease (e.g., a cancerous tumor) may include: anti-cancer agents (e.g., antibodies) and agonistic antibodies that specifically bind to an immunostimulatory receptor, and optionally additional immunostimulatory agents, e.g., antagonistic anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agents (e.g., antibodies), simultaneously or sequentially or any combination thereof, can enhance the anti-tumor immune response of the host.

Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins expressed by tumors that have immunosuppressive effects. These proteins include TGF-. beta. (Kehrl et al (1986) J.Exp.Med.163: 1037-. Antibodies directed against each of these entities may further be combined with agonistic antibodies that specifically bind to an immunostimulatory receptor, with or without additional immunostimulatory agents, such as antagonistic anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agents, such as antibodies, to counteract the effects of the immunosuppressive agent and to favor the anti-tumor immune response of the host.

Other agents (e.g., antibodies) useful for activating the host immune responsiveness may further be used in combination with an agonistic antibody that specifically binds to an immunostimulatory receptor (with or without additional immunostimulatory agents, such as antagonistic anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies). These agents include molecules on the surface of dendritic cells that activate DC function and antigen presentation. The anti-CD 40 antibody (Ridge et al, supra) can be used in combination with an agonistic antibody that specifically binds to an immunostimulatory receptor and optionally an additional immunostimulatory agent (e.g., an anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, such as an antibody). Other antibodies, Weinberge et al, supra, Melero et al, supra, Hutloff et al, supra, which activate T cell co-stimulatory molecules, may also provide increased levels of T cell activation.

As discussed above, bone marrow transplantation is currently being used to treat a variety of hematopoietic tumors. Agonistic antibodies that specifically bind to immunostimulatory receptors, either alone or in combination with CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockade, may be used to increase the effectiveness of donor-transplanted tumor-specific T cells.

Some experimental treatment protocols involve ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells to recipients for antigen-specific T cells against tumors (Greenberg & ridsell, supra). These methods can also be used to activate T cell responses to infectious pathogens such as CMV. In vitro activation in the presence of agonistic antibodies that specifically bind to immunostimulatory receptors (with or without additional immunostimulatory therapy, such as antagonistic anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies) is expected to increase the frequency and activity of adoptively transferred T cells.

Agonistic antibodies that specifically bind to immunostimulatory receptors and combination antibody therapies described herein may be used in combination with (e.g., simultaneously or separately from) additional therapies, such as radiation, chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-FU, or camptothecin + apo2l/TRAIL (6X combination))), one or more proteasome inhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors (e.g., BH3I-2' (Bcl-xl inhibitors), indoleamine dioxygenase-1 inhibitors (e.g., inb 24360, indomod, NLG-919, or F001287), AT-101 (R- (-) -gossypol derivative), ABT-263 (small molecule), GX-15-070 (Obakyrax) or MCL-1 (myeloid leukemia cell differentiation protein-1) antagonist), iAP (inhibitor of apoptotic proteins) antagonist (such as smac7, smac4, small molecule smac mimetics, synthetic smac peptide (see Fulda et al, Nat Med 2002; 8: 808-15), ISIS23722(LY2181308) or AEG-35156(GEM-640))), HDAC (histone deacetylase) inhibitor, anti-CD 20 antibody (such as rituximab), angiogenesis inhibitor (such as bevacizumab), anti-angiogenesis agent against VEGF and VEGFR (such as Avastin), synthetic triterpenoid drug (see Hyer et al, Cancer Research 2005; 4799-.

The agonistic antibodies and combination antibody therapies described herein that specifically bind to immunostimulatory receptors may further be used in combination with one or more antiproliferative cytotoxic drugs. Classes of compounds that may be used as antiproliferative cytotoxic agents include, but are not limited to, the following:

alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazines): urine mustard, nitrogen mustard, Cyclophosphamide (CYTOXAN)^TM) Ifosfamide, melphalan, chlorambucil, triethylenemelamine, triethylenethiocyclophosphamide, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

Antimetabolites (including but not limited to folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, phenastatin and gemcitabine.

Antiproliferative agents, including but not limited to: taxanes, paclitaxel (TAXOL, trade name)^TM) Docetaxel, discodermolide (DDM), Dictyostatin (DCT), phellodendrin A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furan epothilone D, desoxyepothilone B1, [17 ] and]dehydrodesoxyepothilone B, [18 ]]Dehydrodesoxyepothilone B, C12, 13-cyclopropyl epothilone A, C6-C8 bridging epothilone A, trans 9, 10-dehydroepothilone D, cis 9, 10-dehydroepothilone D, 16-demethylepothilone B, epothilone B10, discodermolide, patopilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (clionactone), TZT-1027 (sobiditin), ILX-651 (tasidotin hydrochloride), Halichondrin (Halichondrin) B, eribulin mesylate (E-7389), hemisterlin (Hemiasterlin) (HTI-286), E-74, cryptophycin (cyclins), estrogen-2, maytansine alkaloid (Immunoquinol-1), epothilone B-12, epothilone B-076, epothilone B-12, epothilone B-5, epothilone B-4, epothilone B-O-5, epothilone B-D-1027 (epothilone), epothilone B-6, epothilone B-E-7335, epothilone D-3, epothilone A-5, epothilone A-3, epothilone A-6, epothilone A-5, epothilone A-3, epothilone A-3, and any of which is known in the same or a stable prodrug thereof.

If it is desired to quiesce abnormally proliferating cells concurrently with or prior to treatment with an agonistic antibody that specifically binds to an immunostimulatory receptor, hormones and steroids (including synthetic analogs) may also be administered to the patient, such as 17 a-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drotadalasone propionate, testolactone, megestrol acetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, clorenestrol, hydroxyprogesterone, aminoglutaconeMimette, estramustine, medroxyprogesterone acetate, leuprorelin, flutamide, toremifene, and ZOLADEX^TM. Other drugs that are clinically useful in regulating tumor growth or metastasis, such as antiemetics, may also be administered as needed when using the methods or compositions described herein.

In certain embodiments, an agonist antibody that specifically binds to an immunostimulatory receptor (e.g., an anti-OX 40 antibody such as OX40.21) is administered in combination (simultaneously or separately) with nivolumab to treat a patient having cancer, e.g., colorectal cancer or bladder cancer.

In certain embodiments, an agonist antibody that specifically binds to an immunostimulatory receptor (e.g., an anti-OX 40 antibody, such as OX40.21) is administered in combination (simultaneously or separately) with ipilimumab to treat a patient having a cancer, e.g., ovarian, bladder, or prostate cancer.

Methods for safe and effective administration of chemotherapeutic agents are well known to those skilled in the art. In addition, their methods of administration are also described in the standard literature. For example, methods of administration of many chemotherapeutic agents are described in the Physicians' desk reference (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, N.J.07645-1742, USA); the disclosure of which is incorporated herein by reference.

The chemotherapeutic agent and/or radiation therapy may be administered according to treatment regimens well known in the art. It will be apparent to those skilled in the art that the mode of administration of the chemotherapeutic agent and/or radiation to a disease may vary depending on the disease being treated and the known effects of the chemotherapeutic agent and/or radiation on the disease. Moreover, depending on the treatment regimen administered (e.g., the amount and time of administration), may vary according to the observed effect of the administered therapeutic agent on the patient, and according to the observed response of the administered disease to the administered therapeutic agent, according to the knowledge of the skilled clinician.

Agonistic antibodies binding to immunostimulatory receptors

Agonistic antibodies that bind to immunostimulatory receptors suitable for use in the methods described herein (including combination therapies) include newly developed agonistic antibodies, as well as agonistic antibodies known in the art (including antibodies that compete with or bind to the same epitope as these antibodies). Novel agonistic antibodies targeting immunostimulatory receptors may be obtained using standard antibody production and screening techniques, and agonist activity may be detected using assays well known in the art.

In certain embodiments, suitable agonistic antibodies for use in the methods described herein bind to and activate immunostimulatory receptors and their downstream signaling pathways, thereby stimulating an immune response. In some embodiments, an agonistic antibody that binds an immunostimulatory receptor for use in the methods described herein exhibits a "hook effect," in which the antibody approaches a saturating concentration or a saturating concentration (e.g., a concentration that results in > 80% RO), with reduced efficacy in a functional in vitro assay (e.g., cytokine production, proliferation assay, receptor surface expression) and/or in vivo assay (e.g., anti-tumor efficacy) compared to a concentration that results in less than 80% RO.

In some embodiments, the agonistic antibody binds to a co-stimulatory receptor, for example a co-stimulatory receptor selected from the group consisting of: tumor necrosis factor receptor superfamily members (TNFRSF), ICOS (CD278), CD28, LIGHT, CD40L, TIM1, SLAM, CD1, CD2, CD226, LFA-1(CD11A, CD18), CD5, CD7, CD30, CD54, CD97, CD154, CD160, LIGHT, NKG2C, SLAMF7, NKp80 and TGF-beta. In some embodiments, an agonistic antibody that binds an immunostimulatory receptor used in the methods described herein exhibits a "hook effect.

In some embodiments, the agonistic antibody binds a member of TNFRSF, e.g., a receptor selected from the group consisting of: TNFR1, TNFR2, HVEM, LT beta R, OX40(CD134/TNFRSF4), CD27 (TNFRSF7), CD40, FAS, DCR3, CD30, 4-1BB, TRAILR1, TRAILR2, TRAILR3, TRAILR4, OPG, RANK, FN14, TACI, BAFFR, BCMA, GITR, TROY, DR3 (death receptor 3), DR6 (death receptor 6), XEDAR (xenoprotein A2 receptor), and NGFR. In some embodiments, an agonistic antibody that binds a TNFRSF member for use in the methods described herein exhibits a "hook effect.

In some embodiments, the agonist antibody specifically binds OX40, e.g., an agonist anti-OX 40 antibody that exhibits a "hook effect. Exemplary agonistic anti-OX 40 antibodies are MEDI6469, MEDI0562, PF-04518600, MOXR0916, GSK3174998, RG-7888(von lerolizumab), INCAGN-1949, and WO2016/196228 (e.g., OX 40.21); WO/1995/012673; WO 199942585; WO/2014/148895; WO 15153513; WO 15153514; WO/2013/038191; WO 2016057667; WO 03106498; WO 12027328; WO 13028231; WO 2016200836; WO 17063162; WO 17134292; WO 17096179; WO 17096281; antibodies as described in WO 17096182; the contents of each are hereby incorporated by reference in their entirety. In some embodiments, an agonist anti-OX 40 antibody exhibits the following properties:

(a) binds to membrane-bound human OX40, e.g., with an EC of 1nM or less (e.g., 0.01nM to 1 nM)₅₀E.g., as determined by FACS;

(b) binds to cynomolgus monkey OX40, e.g., binds to membrane-bound cynomolgus monkey OX40, e.g., with an EC of 10nM or less₅₀(e.g., 0.01nM to 10nM), e.g., as determined by FACS;

(c) binds to soluble human OX40, e.g., with a K of 10nM or less_D(e.g., 0.01nM to 10nM), e.g., by

SPR analytical assay

(d) Inducing or enhancing T cell activation as evidenced by (i) increased secretion of IL-2 and/or IFN- γ in OX 40-expressing T cells, and/or (ii) increased T cell proliferation; and

(e) inhibiting the binding of OX40 ligand to OX40, e.g., with an EC of 1nM or less as determined by FACS₅₀For example, in assays using hOX40-293 cells.

In some embodiments, the agonist anti-OX 40 antibody binds to an Fc receptor, e.g., an fey receptor.

In some embodiments, the agonist anti-OX 40 antibody induces or enhances T cell activation by multivalent cross-linking.

In some embodiments, agonist anti-OX 40 antibodies can be produced by activating T_effCellsThe suppression of T effector cells by limiting Treg cells, the depletion and/or suppression of tumor Treg cells and/or the activation of NK cells, e.g. in tumors, to stimulate or enhance an immune response. For example, an agonist anti-OX 40 antibody can activate or co-stimulate Teff cells, e.g., as evidenced by enhanced cytokine (e.g., IL-2 and IFN- γ) secretion and/or enhanced proliferation. In certain embodiments, an agonist anti-OX 40 antibody increases IL-2 secretion by 50%, 100% (i.e., 2-fold), 3-fold, 4-fold, 5-fold or more, optionally up to 10-fold, 30-fold, 100-fold as determined, for example, on primary human T cells or human OX 40-expressing T cells. In certain embodiments, an agonist anti-OX 40 antibody increases IFN- γ secretion by 50%, 100% (i.e., 2-fold), 3-fold, 4-fold, 5-fold or more, optionally up to 10-fold, 30-fold, 100-fold as determined, for example, on primary human T cells or human OX 40-expressing T cells.

In some embodiments, the agonist anti-OX 40 antibody binds to the C1q component of human complement. In some embodiments, the agonist anti-OX 40 antibody induces NK cell-mediated lysis of activated CD4+ T cells. In some embodiments, the agonist anti-OX 40 antibody promotes macrophage-mediated phagocytosis of OX 40-expressing cells. In some embodiments, the agonist anti-OX 40 antibody inhibits regulatory T cell-mediated CD4+ T cell proliferation.

In some embodiments, the agonist anti-OX 40 antibody binds to human OX40 and canine OX40.

In some embodiments, the agonist anti-OX 40 antibody binds all or a portion of sequence DVVSSKPCKPCTWCNLR (SEQ ID NO:15) in human OX40.

In a particular embodiment, the agonist anti-OX 40 antibody used in the methods described herein is OX 40.21. The heavy and light chain sequences, variable region sequences and CDR sequences of OX40.21 are provided below.

TABLE 2 OX40.21 sequence summary

Accordingly, in some embodiments, the anti-OX 40 antibody comprises three variable heavy chain CDRs and three variable light chain CDRs in variable heavy chain SEQ ID NO 11 and variable light chain SEQ ID NO 12, respectively.

In some embodiments, the anti-OX 40 antibody comprises: comprising the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID Nos. 5-7, respectively, and/or the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID Nos. 8-10, respectively.

In some embodiments, the anti-OX 40 antibody comprises heavy chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOS: 5-7, respectively, and/or light chain CDR1, CDR2, and CDR3 sequences consisting of SEQ ID NOS: 8-10, respectively.

In some embodiments, the anti-OX 40 antibody comprises the heavy and light chain variable region sequences set forth in SEQ ID NOs 11 and 12, respectively.

In some embodiments, the anti-OX 40 antibody comprises the heavy and light chain sequences set forth in SEQ ID NOS: 13 and 14, respectively.

Exemplary agonistic antibodies that bind to immunostimulatory receptors include anti-4-1 BB antibodies (e.g., urelumab (BMS-663513) and PF-05082566), anti-GITR antibodies (e.g., TRX518, MK-4166, MK-1248, GWN323, and antibodies disclosed in WO2017087678, the contents of which are herein incorporated by reference in their entirety), anti-CD 27 antibodies (e.g., anti-CD 27 antibodies (e.g., varlillumab (CDX-1127)), anti-ICOS antibodies (e.g., MEDI-570, GSK3359609, JTX-2011), anti-CD 3 antibodies (e.g., ptdr-25), and anti-CD 40 antibodies (e.g., CP-870,893, dacetuzmumab, ChiLob 7/4, lucatumumab, APX005M, ADC-1013, JNJ-64457107, SEA-CD40), and antibodies that compete with and/or bind to and/or the same epitope(s) as these antibodies.

Preferably, the agonistic antibody binds to the immunostimulatory receptor with high affinity, e.g., at 10^-7K of M or less_D，10^-8M or less, 10^-9M or less, 10^-10M or less, 10^-11M or less, 10^-12M or less, 10^-12M to10^-7M，10^-11M to 10^-7M，10^-10M to 10^-7M, or 10^-9M to 10^-7K of M_D。

In some embodiments, the agonistic antibody that binds an immunostimulatory receptor is an antibody selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or variants thereof. In a particular embodiment, the antibody is an IgG1 anti-OX 40 antibody, e.g., OX 40.21.

In certain embodiments, an agonistic antibody that binds an immunostimulatory receptor includes a heavy chain constant region that alters the properties of the antibody. For example, an agonistic antibody may include a modified heavy chain constant region that alters the activity of the antibody relative to an antibody having an unmodified heavy chain constant region. Accordingly, in some embodiments, agonistic antibodies have modifications in the heavy chain constant region that enhance effector function. In other embodiments, the agonistic antibody has a modification in the heavy chain constant region that reduces effector function. Modifications in the Fc region may be used, for example, to (a) increase or decrease antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increase or decrease complement-mediated cytotoxicity (CDC), (C) increase or decrease affinity for C1q, and/or (d) increase or decrease affinity for an Fc receptor, relative to the parent. Specific modifications (e.g., amino acid substitutions) that can be used to generate variant Fc regions having these characteristics are well known in the present invention and are summarized, for example, in WO 2016/196228.

In some embodiments, the agonistic antibody that binds an immunostimulatory receptor is a human, humanized, or chimeric antibody.

In some embodiments, the agonistic antibody that binds an immunostimulatory receptor is a bispecific antibody.

In some embodiments, an agonistic antibody that binds an immunostimulatory receptor is an immunoconjugate conjugated to a moiety such as a detectable label (e.g., a radioisotope, a fluorescent label, an enzyme, and other suitable antibody labels) or an anti-cancer agent (e.g., an antimetabolite, an alkylating agent, a DNA vesicle binding agent, a DNA intercalating agent, a DNA cross-linking agent, a histone deacetylase inhibitor, a nuclear export inhibitor, a proteasome inhibitor, a topoisomerase I or II inhibitor, a heat shock protein inhibitor, a tyrosine kinase inhibitor, an antibiotic, and an anti-mitotic agent). In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

Composition III

The present application also provides compositions, e.g., a pharmaceutical composition, comprising an agonist antibody that binds to an immunostimulatory receptor (e.g., OX40), formulated with a pharmaceutically acceptable carrier for use in methods described herein.

The pharmaceutical compositions described herein may be administered as monotherapy or in combination therapy, for example the combination therapy described herein. For example, combination therapy may include the combined administration of: an agonistic antibody that binds an immunostimulatory receptor, and at least one other anti-cancer agent and/or T cell stimulating (e.g., activating) agent. Examples of therapeutic agents that may be used in combination therapy are described in detail above.

As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the vector is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate or bispecific molecule, may be coated in a material to protect the compound from acids and other natural conditions that might inactivate the compound.

The pharmaceutical compositions described herein may include one or more pharmaceutically acceptable salts. "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not produce any undesirable toxicological effects (see, e.g., Berge, s.m.et al, (1977) j.pharm.sci.66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous, and the like, as well as those derived from non-toxic organic acids such as aliphatic mono-and dicarboxylic acids, benzene-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and the like, as well as those derived from non-toxic organic amines, such as N, N' -dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

The pharmaceutical compositions described herein may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include. (1) Water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogensulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions described herein include: water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. The proper fluidity can be maintained, for example, by: use of coating materials, such as lecithin; in the case of dispersions, the desired particle size is maintained; and the use of surfactants.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization procedures (as described above) and the addition of various antibacterial and antifungal agents, such as parahydroxybenzoic acid, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents, such as sugars, sodium chloride, and the like, may also be added to the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the addition of agents which delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for the preparation of pharmaceutically active substances is known in the present invention. Any conventional media or agent is contemplated unless such media or agent is incompatible with the active compound. The pharmaceutical composition may or may not include a preservative. Supplementary active compounds may be included in the composition.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by: use of coating materials, such as lecithin; in the case of dispersions, the desired particle size is maintained; and the use of surfactants. In many cases, it will be preferable to add isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride to the composition. Prolonged absorption of the injectable pharmaceutical compositions can be brought about by the addition to the compositions of agents which delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared as follows: the active compound is incorporated in the required amount in a suitable solvent together with one or more of the ingredients enumerated above, and then subjected to sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the necessary other ingredients enumerated herein. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yields the active ingredient plus any additional ingredients from a previously filtered solution thereof.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form depends upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of the composition which produces a therapeutic effect. Generally, the amount is generally from about 0.01% to about 99%, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient in 100% in combination with a pharmaceutically acceptable carrier.

The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a bolus may be administered, several divided doses may be administered over a period of time, or the dose may be proportionally reduced or increased as the urgency of the treatment situation dictates. It is particularly advantageous to formulate compositions for parenteral administration in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form (dosage unit form) as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms described herein are dependent upon and directly depend upon (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the inherent limitations of the prior art for compounding such sensitivity for use in treating an individual. Suitable doses of an agonist antibody that binds to an immunostimulatory receptor (e.g., an anti-OX 40 antibody) can be determined using the methods described herein.

For administration of an anti-OX 40 antibody (e.g., OX40.21), the dose ranges from about 0.0001 to 100mg/kg body weight, about 0.01 to 10mg/kg body weight, about 0.01 to 5mg/kg body weight, about 0.1 to 1mg/kg body weight, about 0.1 to 0.5mg/kg body weight, or about 0.5 to 0.8mg/kg body weight. For example, the dose may be 0.2mg/kg body weight, 0.3mg/kg body weight, 0.5mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, or 10mg/kg body weight, or in the range of 1-10mg/kg body weight. In certain embodiments, the dose is 0.2 mg/kg. In certain embodiments, the dose is 0.25 mg/kg. In other embodiments, the dose is 0.5 mg/kg.

In certain embodiments, for combination therapy with an anti-OX 40 antibody and an anti-PD-1 or anti-CTLA-4 antibody, the administration can be at a fixed dose. Accordingly, in certain embodiments, the anti-OX 40 antibody is administered at a fixed dose of about 25 to about 320mg, e.g., at a fixed dose of about 25 to about 160mg, about 25 to about 80mg, about 25 to about 40mg, about 40 to about 320mg, about 40 to about 160mg, about 40 to about 80mg, about 80 to about 320mg, about 30 to about 160mg, or about 160 to about 320 mg. In one embodiment, the anti-OX 40 antibody is administered at a dose of 20mg or about 20 mg. In another embodiment, the anti-OX 40 antibody is administered at a dose of 40mg or about 40 mg. In another embodiment, the anti-OX 40 antibody is administered at a dose of 80mg or about 80 mg. In another embodiment, the anti-OX 40 antibody is administered at a dose of 160mg or about 160 mg. In another embodiment, the anti-OX 40 antibody is administered at a dose of 320mg or about 320 mg. An exemplary treatment regimen includes administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 4 months, or once every 3 to 6 months.

In some embodiments, the anti-PD-1 antibody is administered at a fixed dose of about 100 to 300 mg. For example, the dose of the immunooncology agent may be 240mg or about 240mg, 360mg or about 360mg, or 480mg or about 480 mg. In certain embodiments, the dose of anti-PD 1 antibody ranges from about 0.0001 to about 100mg/kg, more typically from about 0.01 to about 5mg/kg of host body weight. For example, the dose may be 0.3mg/kg body weight or about 0.3mg/kg body weight, 1mg/kg body weight or about 1mg/kg body weight, 3mg/kg body weight or about 3mg/kg body weight, 5mg/kg body weight or about 5mg/kg body weight, or 10mg/kg body weight or about 10mg/kg body weight, or in the range of 1-10mg/kg body weight. In some embodiments, the dose of the anti-PD-1 antibody is 240mg or about 240mg administered once every 2 weeks (Q2W). The dose may be scaled (at a dose of 120mg per week) for longer or shorter periods, for example 360mg once every 3 weeks (Q3W) or 480mg once every 4 weeks (Q4W).

In some embodiments, for combination therapy with an anti-OX 40 antibody and an anti-PD-1 antibody, the antibodies can be administered at a fixed dose. In one embodiment, the anti-OX 40 antibody and the anti-PD-1 antibody are administered for at least one administration cycle, wherein the cycle is a 12-week period, wherein in each cycle of the at least one cycle, at least one dose of the anti-OX 40 antibody is administered at a fixed dose of about 1,3, 10, 20, 40, 50, 80, 100, 130, 150, 180, 200, 240, or 280mg and at least 3 doses of the anti-PD-1 antibody are administered at a fixed dose of about 50, 80, 100, 120, 150, 180, 200, 240, 480, 720, or 960 mg. In one embodiment, for each cycle of the at least one cycle, 1 dose of the anti-OX 40 antibody is administered at a fixed dose of about 20, 40, or 80mg and 3 doses of the anti-PD-1 antibody are administered at a fixed dose of about 480 mg. In one embodiment, for each cycle of the at least one cycle, 1 dose of the anti-OX 40 antibody is administered at a fixed dose of about 20mg and 3 doses of the anti-PD-1 antibody are administered at a fixed dose of about 480 mg. In one embodiment, for each cycle of the at least one cycle, 1 dose of the anti-OX 40 antibody is administered at a fixed dose of about 40mg and 3 doses of the anti-OX 40 antibody are administered at a fixed dose of about 480 mg. In one embodiment, for each cycle of the at least one cycle, 1 dose of the anti-OX 40 antibody is administered at a fixed dose of about 80mg and 3 doses of the anti-PD-1 antibody are administered at a fixed dose of about 480. In one embodiment, the anti-PD-1 antibody is administered on days 1, 29, and 57 of each cycle. In one embodiment, the anti-OX 40 antibody is administered on day 1 of each cycle. In one embodiment, the anti-PD-1 antibody is administered on days 1, 29, and 57 of each cycle and the anti-OX 40 antibody is administered on day 1 of each cycle. In one embodiment, the 12 week administration cycle may be repeated as desired. In one embodiment, administration consists of up to 9 cycles. In one embodiment, administration consists of 1, 2,3, 4,5, 6, 7, 8, or 9 cycles. In one embodiment, the anti-OX 40 antibody comprises heavy chain CDR1, CDR2, and CDR3 sequences, wherein the heavy chain CDR1, CDR2, and CDR3 sequences comprise SEQ ID NO's 5-7, respectively, and light chain CDR1, CDR2, and CDR3 sequences, wherein the light chain CDR1, CDR2, and CDR3 sequences comprise SEQ ID NO's 8-10, respectively. In one embodiment, the OX-40 antibody comprises OX 40.21. In one embodiment, the anti-PD-1 antibody comprises nivolumab.

In some embodiments, the anti-CTLA-4 antibody is administered at a dose of about 0.1mg/kg to about 10 mg/kg. For example, the dose may be 1mg/kg or about 3mg/kg of host body weight.

In certain embodiments, the anti-OX 40 antibody is administered prior to the anti-PD-1 or anti-CTLA-4 antibody when administered on the same day. In certain embodiments, the anti-OX 40 antibody is administered after the anti-PD-1 or anti-CTLA-4 antibody when administered on the same day. In certain embodiments, the anti-OX 40 antibody is administered simultaneously with the anti-PD-1 or anti-CTLA-4 antibody when administered on the same day.

In some cases, two or more monoclonal antibodies with different binding specificities are administered simultaneously, such that the dose of each antibody is within the above range. Furthermore, antibodies are typically administered in a number of situations. The interval between single administrations can be, for example, weekly, monthly, every three months, or yearly. The intervals may also be irregular, as suggested by measuring the blood concentration of the target antigen antibody in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of about 1-1000 μ g/ml, and in some methods, the dose is adjusted to achieve a plasma antibody concentration of about 25-300 μ g/ml.

The antibody may be administered in a sustained release formulation, in which case less frequent administration is required. The dosage and frequency of administration will vary with the half-life of the antibody in the patient. In general, human antibodies exhibit the longest half-life, followed in turn by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration may vary depending on whether prophylactic or therapeutic treatment is employed. In prophylactic applications, administration is carried out at relatively low doses, relatively infrequent intervals, over a longer period of time. Some patients will continue to receive treatment for the remainder of their life. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until progression of the disease is reduced or terminated, preferably until the patient exhibits partial or complete remission of the disease symptoms. Thereafter, the patient may be subjected to a prophylactic treatment regimen.

The actual dosage level of the active ingredient in the pharmaceutical compositions described herein can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors including: the activity of a particular composition described herein or an ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the particular composition employed, the age, sex, body weight, condition, general health and past medical history of the patient being treated, and like factors well known in the medical arts.

The "therapeutically effective dose" of the antibody preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic phases of the disease, or prevention of damage or disability due to disease affliction. In the context of cancer, a therapeutically effective dose preferably results in an increase in survival rate, and/or prevents further worsening of the physical symptoms associated with the cancer. Symptoms of cancer are well known in the present invention and include, for example, unusual nevus characteristics, changes in the appearance of the nevus, including asymmetry, changes in borders, color and/or diameter, newly pigmented skin areas, abnormal nevus, subungual black areas, breast bumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, eating difficulties, loss of appetite, chronic cough, increased dyspnea, hemoptysis, bloody urine, bloody stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal distension, abdominal fullness, abdominal distension, abdominal dropsy, vaginal bleeding, constipation, abdominal distension, colonic perforation, acute peritonitis (infection, fever, pain), pain, hematemesis, profuse sweat dribbles, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, muscle spasms, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, cold stroke, and the like, Colon metastasis, lung metastasis, bladder metastasis, liver metastasis, bone metastasis, kidney metastasis, and pancreas metastasis, dysphagia, and the like.

A therapeutically effective dose may prevent or delay the onset of cancer, which may be desirable, for example, when early or preliminary signs of disease are present. Laboratory tests used in cancer diagnosis include chemistry, hematology, serology, and radiology. Accordingly, any clinical or biochemical assay that can monitor any of the foregoing can be used to determine whether a particular therapeutic is an effective dose for treating cancer. One of ordinary skill in the art will be able to determine such amounts depending on the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected, among other factors.

The antibodies and compositions described herein can be administered by one or more routes of administration using one or more of a variety of methods known in the art. One skilled in the art will appreciate that the route and/or mode of administration will vary depending on the desired result. Preferred routes of administration of the antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, the antibodies of the invention may be administered by a non-parenteral route, for example by a topical, epidermal or mucosal route of administration, for example, intranasal, oral, vaginal, rectal, sublingual or topical.

The active compounds may be formulated with carriers that protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated administration systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyethylene oxide, and polylactic acid. Many methods of preparing such formulations are patented or are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

The antibody composition may be administered using medical devices known in the present invention. For example, in one embodiment, the composition is administered using a needleless hypodermic injection device, such as, for example, U.S. Pat. nos. 5,399,163; 5,383,851, respectively; 5,312,335, respectively; 5,064,413, respectively; 4,941,880, respectively; 4,790,824, respectively; or 4,596,556. Examples of well-known implants and modules for antibody administration include: U.S. patent No. 4,487, 603, which discloses an implantable micro-infusion pump for dispensing a drug at a controlled rate; U.S. patent No. 4,486,194, which discloses a therapeutic device for administering a drug through the skin; U.S. patent No. 4,447,233, which discloses a medication infusion pump for delivering a medication at a precise infusion rate; U.S. patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multiple lumen compartments; and us patent No. 4,475,196, which discloses an osmotic pressure drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, agonistic antibodies that bind to immunostimulatory receptors are formulated to ensure their proper distribution in vivo. For example, the Blood Brain Barrier (BBB) can block many highly hydrophilic compounds. To ensure that the antibody is able to cross the BBB (if desired, e.g., for brain cancer), it may be formulated, for example, as a liposome. For methods of making liposomes, see, e.g., U.S. Pat. nos. 4,522,811; 5,374,548, respectively; and 5,399,331. Liposomes can include one or more specific molecular moieties that are selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, e.g., v.v. ranade (1989) j.clin.pharmacol.29: 685). Exemplary targeting molecule moieties include: folic acid or biotin (see, e.g., U.S. patent No. 5,416,016 to Low et al); mannoside (Umezawa et al, (1988) biochem. biophysis. Res. Commun.153: 1038); antibodies (P.G.Blueman et al, (1995) FEBSLett.357: 140; M.Owais et al (1995) antibodies.Agents Chemother.39: 180); the surfactant protein a receptor (Briscoe et al, (1995) am.j. physiol. 1233: 134); p120(Schreier et al (1994) J.biol.chem.269: 9090); see also k, keenanen; M.L.Laukkanen (1994) FEBSLett.346: 123; j.j.killion; fidler (1994) Immunomethods 4: 273.

Kits and unit dosage forms

The invention also provides kits comprising a pharmaceutical composition comprising an agonist antibody that binds to an immunostimulatory receptor (e.g., an anti-OX 40 antibody), in an amount determined using the methods described herein, and a pharmaceutically acceptable carrier, for use, e.g., in treating cancer. The kit can further comprise at least one additional agent (e.g., an agent suitable for the combination therapy described herein, such as another therapeutic agent). The kits of the invention also optionally include instructions, e.g., including an administration schedule, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the compositions contained therein to a patient having a cancer (e.g., a solid tumor). The composition may also include a syringe.

Alternatively, the kit of the invention comprises multiple packages of single dose pharmaceutical compositions, each containing an effective amount of the antibody for one administration according to the methods provided above. The apparatus or devices required for administration of the composition may also be included in the kit. For example, the kit may provide one or more pre-filled syringes containing an amount of antibody.

The kit typically includes a label indicating the intended use of the contents of the kit. The term "label" includes any written or recorded material provided on, with, or otherwise accompanying the kit.

Examples

OX-40 is a co-stimulatory receptor that is upregulated upon T cell activation. It increases CD4⁺And CD8⁺Activation, proliferation and survival of effector T cells (Teff) while suppressing suppression of T cells (tregs). Described herein is an example of a fully human IgG1 agonist monoclonal antibody that binds OX-40 with high affinity and enhances Teff increaseReproduction and inhibition of Treg suppression. Figure 30 provides a depiction of the mechanism of action of the agonist monoclonal antibodies with OX-40.

Example 1 anti-tumor Activity of different doses of ligand-blocking and ligand-non-blocking anti-OX 40 antibodies

This example describes the effect of different doses of OX40.23-mIgG1 (ligand blocking agonist antibody) and OX40.3-mIgG1 (non-ligand blocking agent) in a CT26 mouse tumor model.

BalbC mice implant 1 × 10⁶And CT26 cells. Established CT26 tumors (75-150 mm) were treated on day 6 post-implantation³) Mice of (3) were treated with OX40.23 mIgG1 or OX40.3 mIgG1 at doses of 0.03, 0.1, 0.3, 1,3 and 10mg/kg (Q7D × 2, d 6).

Mean and median tumor growth curves for each treatment group are shown in fig. 1A (IgG control group and OX40.23) and fig. 1B (OX40.3), with mean and median tumor volumes for OX40.23-mIgG1 and OX40.3-mIgG1 treated mice shown in fig. 2A-2D. The mean and median tumor volume, TGI, and the number of tumor-free mice at the end of the study for each treatment group are summarized in table 3.

TABLE 3 tumor volume, TGI and number of tumor-free mice of OX40.23 and OX40.3 as monotherapies are summarized by treatment group.

Abbreviations: TGI, tumor growth inhibition; TV, tumor volume

OX40.3 was administered at 1mg/kg, 3mg/kg and 10mg/kg and showed significant tumor suppression at day 20 compared to isotype control with median TGI of 76%, 93% and 98%, respectively, and median tumor-free mice at the end of the study (day 66) of 1/10, 2/10 and 6/10, respectively. OX40.23, administered at 0.3mg/kg, 1mg/kg, 3mg/kg, and 10mg/kg, showed significant tumor inhibition at day 20, resulting in 59%, 91%, 88%, and 73% median TGI, compared to the isotype control group, and tumor-free mice at the end of the study (day 66) were 2/10, 1/10, 3/10, and 6/10, respectively. Although all groups receiving OX40.23 at doses above 0.3mg/kg had reduced tumor volume, the activity of the 10mg/kg treatment was lower compared to 1mg/kg and 3 mg/kg. This reduction in antitumor activity at high dose (10mg/kg) ("hook" effect) was not observed in the OX40.3 treated group, which showed the greatest TGI and the greatest tumor-free mice at the highest dose in the OX40.3 treated group. Given that OX40.23 blocks OX40-OX40L interactions while OX40.3 does not, these data suggest that the involvement of OX40L may contribute to a reduction in the anti-tumor efficacy of OX40.23 at high doses.

Example 2: alteration of anti-tumor efficacy of anti-OX 40 antibody + anti-PD 1 antibody combination therapy

This example describes the effect of varying doses of OX40.23-mIgG1 used in combination with anti-PD 1 antibody.

The experimental conditions were substantially the same as those described in example 1. The individual tumor growth curves of OX40.23(Q7D x 2) + anti-PD 1 antibody (IgG 1D 265A; Q4D x 3) treated mice are shown in FIGS. 3A and 3B, while the curves of OX40.3+ anti-PD 1 antibody treated mice are shown in FIG. 4. Table 4 summarizes mean and median tumor volumes, TGI and number of Tumor (TF) -free mice per treatment group at the end of the study for OX40.23+ anti-PD 1 treated mice, and table 5 summarizes mean and median tumor volumes, TGI and number of tumor-free (TF) -free mice at the end of the study for OX40.3+ anti-PD 1 treated mice.

TABLE 4 summary of tumor volume, TGI and tumor-free mouse fraction treatment groups of OX40.23 and OX40.3 in combination with anti-PD-1 antibodies

TABLE 5 tumor volume, TGI and number of tumor-free mice of OX40.3 in combination with anti-PD-1 antibodies are summarized by treatment group.

As shown in Table 4, OX40.23 showed significant improvement in antitumor efficacy at doses ranging from 0.1mg/kg to 3mg/kg when administered in combination with anti-PD 1 antibody, with a tumor-free rate of over 80% at the end of the study, compared to 1/10 for the anti-PD-1 antibody monotherapy. However, at the highest dose of OX40.23(10mg/kg), both mean and median tumor volumes were higher than in the low dose group, with only 6 of 10 mice being tumor-free at the end of the study, indicating a reduction in antitumor activity at the high dose, similar to that observed in OX40.23 monotherapy in example 1 ("hook" effect).

The effective dose of OX40.23-mIgG1 in combination with anti-PD 1 antibody is 0.1-0.3mg/kg, which is about 1/10 of the effective dose (1-3mg/kg) of OX40.23-mIgG1 monotherapy.

As shown in table 5, when OX40.3 was used in combination with anti-PD 1 antibody at doses of 3mg/kg and 10mg/kg, 8/10 and 7/10 TF mice, respectively, at the end of the study (day 124) inhibited the growth of CT26 tumors more strongly than anti-PD 1 antibody alone, with a median TGI of over 70% at day 19; in contrast, the median TGI of the anti-PD 1 antibody monotherapy was 4/10 TF mice at day 124 and 32% at day 19. Similar fold changes in efficacy at lower doses were also observed when OX40.23 was used in combination with anti-PD 1 antibody, as with OX40.23 (figure 4).

Example 3 anti-tumor Activity of anti-OX 40 and anti-PD 1 antibodies administered simultaneously or consecutively

This example describes the comparison of simultaneous administration of an anti-OX 40 antibody and an anti-PD 1 antibody to the anti-tumor activity of sequential administration.

The mouse tumor model used in this experiment was essentially as described in examples 1 and 2. OX40.23 was administered to mice at a dose of 0.03mg/kg, 0.3mg/kg, or 3mg/kg on days 5 and 12 after tumor implantation, followed by concurrent administration of anti-PD-1 on days 5,9, and 13, or delayed administration of anti-PD-1 on days 10, 14, and 18, the latter providing sequential treatment.

As shown in figure 5, due to the rapid progression of CT26 tumor, anti-PD-1 antibody alone delayed treatment starting on day 10 showed lower activity compared to treatment starting on day 5, the former did not have tumor regression, and the latter resulted in 3 out of 10 mice being tumor-free. Concurrent treatment of OX40.23 plus anti-PD-1 antibody resulted in a significant improvement in antitumor activity, with 8/10, 6/10, and 10/10 mice, respectively, being tumor-free at 0.03mg/kg, 0.3mg/kg, and 3mg/kg of OX 40.23. The antitumor activity of OX40.23 plus delayed anti-PD-1 was similar to that of the concurrent treatment, with no tumor mice being 6/10 and 7/10 at 0.3mg/kg and 3mg/kg of OX40.23, respectively. Combination of 0.03mg/kg of OX40.23 with delayed anti-PD-1 treatment did not show better anti-tumor activity compared to the corresponding OX40.23 or anti-PD-1 monotherapy. These results indicate that simultaneous administration is similar to sequential administration in terms of antitumor activity.

Furthermore, anti-OX 40 agonist in combination with either anti-PD-1 or anti-CTLA-4 was tested in the same mouse tumor model used in this experiment, essentially as described in examples 1 and 2. BMS-986178 was administered to mice either alone or in combination with anti-PD 1 or anti-CTLA.

As shown in FIG. 38, anti-OX 40 agonist administered in combination with anti-PD-1 or anti-CTLA-4 showed enhanced anti-tumor activity. Of the 10 mice treated with anti-PD-1 and BMS-986178, 10 mice were tumor-free, and of the 10 mice treated with anti-CTLA-4 and BMS-986178, 8 mice were tumor-free.

Examples 1-3 in summary, OX40.23 (a ligand blocking, agonistic antibody) reached maximum activity at 3mg/kg in monotherapy; maximum activity was achieved at 0.3mg/kg when treated in combination with anti-PD 1 antibody. Similar fold changes in dose lowering were also observed for the efficacy of the ligand non-blocking antibody OX 40.3: in monotherapy, maximal activity is achieved at 10 mg/kg; in combination therapy with anti-PD-1 antibodies, maximal activity was achieved at 3 mg/kg. In both monotherapy and combination therapy, a "hook" effect (reduced activity) was observed when OX40.23 was administered at 10 mg/kg. However, this "hook" effect was not observed in mice treated with OX40.3 alone or in combination with anti-PD-1 antibodies, indicating that the reduction in efficacy observed with OX40.23 at higher doses is dependent on the interaction of OX40L-OX 40. Furthermore, for combination therapy, the antitumor activity resulting from simultaneous administration is comparable to staggered (stagered) dosing regimens.

Example 4: receptor occupancy of OX40.23 as a single agent or administered in combination with an anti-PD 1 antibody

To better understand the potential mechanism of the "hook" effect in anti-tumor efficacy observed when anti-OX 40 antibody was administered at high doses (10mg/kg) in mice either monotherapy or in combination with anti-PD 1 antibody, OX40 Receptor Occupancy (RO) and OX40 expression on CD4+ T cells in the blood and tumor microenvironment was evaluated.

The dosing and sampling schedule is shown in FIG. 6A briefly, BalbC mice were implanted with 1 × 10⁶And CT26 cells. On day 6 post-implantation, established CT26 tumors (75-150 mm) were treated at the indicated doses³) Is administered at a dose of 10mg/kg, wherein the anti-PD 1 antibody is administered.

Receptor occupancy in tumors and blood was evaluated at day 8 and day 13 post-tumor implantation to assess the kinetic changes in OX40 receptor occupancy and OX40 surface expression in the CD4 subpopulation resulting from OX40.23 monotherapy and combination therapy with anti-PD-1 antibodies. Blood was collected by cardiac puncture into a syringe containing ethylenediaminetetraacetic acid (EDTA). By passing

(Sigma-Aldrich) gradient separation method to recover viable leukocytes. 2ml of Histopaque-1083 was added to a 15ml conical centrifuge tube and anticoagulated whole blood was carefully layered onto the tissue protein medium. During centrifugation, erythrocytes and neutrophils are aggregated by ficoll and rapidly sedimented. PBMC were resting on the plasma-Histopaque 1083 interface. Most of the extraneous platelets were removed by low speed centrifugation in a washing step. Tumors were removed, weighed, and placed in a generic MACS Octon Dispmagnet^TM(Miltenyi) on-line; tumor treatment a mouse tumor dissociation kit (Miltenyi) was used. After dissociation, the cell suspension was washed, filtered and counted.

Single cell suspensions from blood and tumors of a single mouse were made in duplicate on two plates and used to test receptor occupancy and total receptors, respectively. To test for total receptors, an excess of OX 40.23-biotin antibody prepared in FACS buffer (2% FBS and 2mM EDTA in PBS) was added to a final concentration of 10g/ml and stained for 30 min at 4 ℃. The samples were then washed 3 times with FACS buffer and then stained with PE-streptavidin at a concentration of 0.5g/ml for 30 minutes. To test for occupied receptors, only PE-streptavidin was added and stained at 0.5g/ml for 30 minutes. Then, both the total and occupied samples were washed three times and stained for immune cell markers with flow cytometry antibodies. Before flow cytometry, 1. mu.g/ml of DAPI was added to distinguish between live and dead cells. Antibody fluorescence was detected by flow cytometry on fortessa (bd biosciences) and the results were analyzed using FlowJo software (FlowJo, LLC). Receptor occupancy was calculated for each animal according to the following formula: % RO [ ([ "test" Δ MFI ]/[ "total" Δ MFI ]) x100, where "test" is the amount of receptor occupied by OX40.23 when assessed directly in ex vivo and "total" is the total amount of receptor determined from the amount of excess OX 40.23-biotin added to the sample.

As shown in fig. 6B, receptor occupancy was dependent on antibody dose, with similar receptor occupancy observed at higher doses in tumor and peripheral blood. Receptor occupancy rates are similar, particularly between monotherapy and combination therapy. Furthermore, a decrease in the occupancy of OX40 receptors in peripheral blood at day 13, but an increase in the occupancy in tumors, particularly at 3mg/kg and 10mg/kg doses, suggests clearance of anti-OX 40 antibody in blood and accumulation of anti-OX 40 antibody in tumors, compared to day 8.

To further investigate the modulation of OX40 receptor occupancy, the total and occupancy levels of OX40 receptor were independently assessed. Throughout the dose escalation process, the fraction of OX40 receptor that was occupied on the cell surface continued to increase, and no significant difference was shown between day 8 and day 13 (FIG. 6C). However, at doses of 0.3mg/kg and above (receptor occupancy of 0.3mg/kg is approximately 40%), the total amount of OX40 receptors on the cell surface showed a rapid downregulation at day 8 (two days post-dose) and remained low until day 13 of the study. These data suggest that the high calculated receptor occupancy observed at 3mg/kg and 10mg/kg doses of OX40.23 is likely driven by down-regulation of total OX40 receptors, rather than by an increase in the amount of occupied receptors.

The relationship between percent tumor growth inhibition and OX40 receptor occupancy shows that for the combination of OX40.23+ anti-PD 1, maximum tumor growth inhibition was achieved at receptor occupancy of 20-80%, while tumor growth inhibition decreased when OX40 receptor occupancy exceeded 80% (fig. 6D).

In a similar experiment, CT26 tumor mice were treated with BMS-986178 or isotype antibody IgG control. At designated time points post-treatment, tumor samples were assessed for OX40RO and total surface OX40 by flow cytometry.

As shown in FIGS. 34A-34D, tumor samples from CT26 tumor-bearing mice showed a dose-dependent correlation of OX40RO on tumor Tregs with BMS-986178 7 days after BMS-986178 treatment (FIG. 34A), while total OX40 expression on tumor Tregs decreased at BMS-986178 doses ≧ 0.3mg/kg (FIG. 34B). Furthermore, as the mAb occupancy of OX40 decreased over time (day 10 and later after BMS-986178 treatment, fig. 34C), surface expression of OX40 recovered (fig. 34D). It can be seen that in the CT26 mouse model, the expression of OX40 on tumor Tregs decreased with increasing dose of anti-BMS-986178.

Taken together, these data indicate that the therapeutic efficacy of the agonist anti-OX 40 antibody in combination with anti-PD 1 antibody is significantly improved compared to anti-PD 1 antibody monotherapy, which achieves maximal anti-tumor activity at OX40 receptor occupancy rates well below 100%. Deep downregulation of surface OX40 was observed within 2 days post antibody administration when receptor occupancy approached around 40%, with the downregulation being maintained at least until 7 days post-administration. This down-regulation of OX40 might explain why the therapeutic activity of this combination was lower at the anti-OX 40 dose of 10 mg/kg. It follows that the appropriate choice of antibody dose and frequency of application is required to minimize or prevent the reduction in anti-tumor activity of agonist anti-OX 40 antibodies at high doses (e.g., 10 mg/kg).

Example 5 receptor occupancy, surface expression of OX40, and drug Exposure in human patients administered OX40.21

This example demonstrates the correlation between the loss of surface expression of OX40 with increasing receptor occupancy in human patients.

Receptor occupancy and surface OX40 expression in CD4+ T cells or Tregs was determined in human patients administered OX40.21 (an agonist anti-OX 40 antibody) 20mg, 40mg, 80mg, 160mg or 320mg using the methods described in example 4 (the "test" vs. "total" equation for determining RO is the same as in example 4, but modified to be suitable for use in calculating OX40.21 RO in human patients). Receptor occupancy in CD4+ T cells and Tregs was between 80 and 100% for each dose cohort, both at cycle 1 day 8 and cycle 2 day 1. As shown in fig. 7A, and consistent with the results of example 4, in human patients, the surface expression of OX40 tended to decrease as the receptor occupancy of OX40.21 increased. Similar receptor occupancy was also observed in peripheral blood of each cohort as with CD4+ T cells and Tregs, with > 80% receptor occupancy observed at doses >20mg and a plateau following the first dose (C2D1) in the second cycle.

In terms of drug exposure, as shown in figure 7B, OX40.21 showed a linear PK when administered in combination with nivolumab, with dose-related increases in exposure in the 20-320mg range.

Example 6: PK-PD model in an alternative administration plan for predicting receptor occupancy

This example describes a PK-PD model for predicting receptor occupancy in human patients. Drug concentration-time profiles were described by a linear two-compartment PK model using population PK analysis. As shown in fig. 8A and fig. 40, this PPK model appears to provide a reasonable description of the observed concentration data. Individual drug exposures (Cmin 1: trough concentration after the first dose) in an alternative dosing schedule were predicted from this PPK model for subsequent PK-PD model development. Based on RO data for 16 cancer patients administered OX40.21 in the 20-320mg dose range, an Emax model (the following equation) was used to describe the relationship between drug concentration (Cmin 1: trough concentration after the first dose) and blood RO of C2D 1.

Wherein the "drug concentration" is Cmin1, trough concentration after the first dose, RO_maxMaximum percentage of blood RO, EC₅₀To correspond to RO _max50% of the drug concentration. Based on this analysis, drug EC was estimated₅₀It was 0.094. mu.g/ml (FIG. 8B). Using the established PK-PD relationship (Emax model), taking into account PK and PD variability among subjects, experimental stimulation was performed to predict blood RO under an alternative dosing schedule (fig. 9).

Tumor RO was also predicted (fig. 10A and 10B), based on data generated from human tumor biopsy samples (N ═ 5) of different cancer types (head and neck, cervical, urothelial, and colorectal), measuring total drug and total OX40 concentrations in tumor homogenates at OX40.21 doses of 20-320 mg. To predict tumor RO at different doses and dosing schedules, the following assumptions were made: 1) EC50 for tumor RO was the same as in serum; 2) the level of tumor drug is in equilibrium with the level of drug in serum; 3) The mean value of the observed tumor to serum concentration ratio was 0.20 (range 0.07-0.47); 4) the observed levels of OX40 averaged 0.15nM (range 0.02-0.47 nM). Consistent with the context used, the blood RO of the tumor RO (fig. 10A) was predicted. However, when tumor RO prediction was performed using a high OX40 level of 0.47nM, and a low tumor drug penetration ratio of 0.07, tumor RO deviated from blood RO (fig. 10B).

Model-based simulation results show that exposure to OX40.21 doses of 40mg q4w, 40mg q8w, 40mg q12w, and 80mgq12w may result in a wider range of receptor occupancy in blood (e.g., RO about 20% to about 90%, fig. 9), as well as in tumors (e.g., RO about 10% to about 90%, at an average target level of 0.15nM and an average tumor penetration ratio of 0.20, fig. 10A and 10B), and thus may provide an opportunity to see if equivalent or even superior PD responses can be achieved at lower dosing frequencies.

Example 7 in vitro assessment of the relationship between OX40.21 concentration and receptor occupancy

This example describes the assessment of receptor occupancy in T cells in vitro based on different concentrations of OX 40.21.

Briefly, total T cells were purified from human whole blood using Ficoll gradient centrifugation. CD4+ T cells were isolated from PBMCs using the Miltenyi CD4+ isolation kit. Isolated T cells were cultured in the presence of irradiated CHO-OKT3-CD32A (artificial antigen presenting cells) and serially diluted OX40.21 or isotype controls. The ratio of bound OX40 antibody to total surface OX40 was determined by flow cytometry as receptor occupancy. Bound antibody was assessed by adding fluorescently conjugated anti-human Fc after T cell washing. Total OX40 was determined by adding saturating amounts of OX40 antibody to T cells. After incubation, cells were washed and stained by adding fluorescently conjugated anti-human Fc.

As shown in fig. 11A and 11B, complete receptor occupancy correlates with down-regulation of total surface OX40 (at OX40.21 >1 nM), reflecting a "hook" effect. Increasing OX40.21 concentrations also induced OX40 on the surface of CD4+ T cells, and higher concentrations down-regulated OX40. The time course of OX40 surface expression showed that the "hook" effect induced by OX40.21 correlated with the highest level of OX40 expression at day 4 (fig. 12A (isotype control) and fig. 12B (OX 40.21))).

To determine whether this effect is specific to OX40.21, the effect of CD28 antibody on surface OX40 levels was also assessed. Briefly, total T cells were purified from human whole blood using Ficoll gradient centrifugation. CD 4T cells were isolated from PBMCs using the Miltenyi CD4+ isolation kit. Isolated T cells were cultured in the presence of irradiated CHO-OKT3-CD32A (artificial antigen presenting cells) and serially diluted agonist anti-OX 40 antibody, isotype control or anti-CD 28 antibody (clone CD 28.2). Total surface OX40 was determined by flow cytometry as receptor occupancy with bound OX40 antibody. Bound antibody was assessed by adding fluorescently conjugated anti-human Fc after washing T cells. Total OX40 was determined by adding saturating amounts of anti-OX 40 antibody to T cells. After incubation, cells were washed and stained by adding fluorescent conjugated anti-human Fc.

As shown in figures 13A, 13B, 32A and 32B, co-stimulation of T cells with anti-CD 28 antibody induced high levels of OX40 expression without loss of expression at higher concentrations, showing that the effect of OX40.21 on overall surface OX40 expression is specific to OX 40.21. Here, monoclonal antibodies against CD137 (fig. 13A and 32A) and CD28 (fig. 13B and 32B) did not show down-regulation of total surface OX40 associated with the "hook" effect. Here, no loss of OX40 surface expression was observed when CD4+ T cells were treated with other costimulatory agonist antibodies.

Further experiments were performed to assess interferon-gamma (IFN- γ) secretion, a quantitative marker of T cell activation (readout), and its relationship to receptor occupancy. The experimental conditions were the same as above except that the supernatant was collected 4 days after culturing the cells. Quantitative measurements were made of IFN-. gamma.secretion using standard ELISA (BD biosciences) or homogeneous time-resolved fluorescence (HTRF) (Cisbios).

As shown in FIG. 14, OX40.21 induced T cell activation as reflected by secretion of IFN- γ. The largest IFN- γ was obtained at approximately 20% receptor occupancy. IFN-gamma secretion is reduced at 100% receptor occupancy, consistent with a "hook" effect.

The relationship between T cell proliferation and receptor occupancy was also evaluated. The experimental conditions were the same as above except for the following: after culturing the cells for 4 days, the cells were spiked for 16 hours by adding 3[ H ] -thymidine and scintillation was counted to test cell proliferation.

As shown in FIGS. 15A-15C, OX40.21 induced T cell proliferation, with maximal proliferation achieved at receptor occupancy between 20-50%. T cell activation (measured by geometric mean fluorescence intensity of CD25 [ GMFI ] on CD4+ T cells) achieved activation of T cells at the lowest dose of BMS-986178 and plateaus at 40% OX40 RO. FIG. 31. Taken together, these data indicate that the maximum activity of OX40.21, in terms of OX40 surface expression, T cell proliferation, and cytokine production, is obtained from receptor occupancy rates of around 20%. At 100% receptor occupancy, a "hook" effect is observed, with reduced functionality/activation of T cells.

Further experiments were performed to assess the suppression of Tregs in BMS-986178 treated T cells. Human CD4+ T cells were differentiated into Tregs and expanded using Dynabeads (α CD3/α CD 28; Thermo Fisher Scientific), rapamycin, and Interleukin (IL) -2. Figure 33A depicts a Treg suppression assay. The Treg phenotype was confirmed by flow cytometry as described herein. Purified CD4+ T cells and CD8+ T cells were cultured under conditions of ± Treg (5:1) + BMS-986178. IL-2 secretion was then determined by a bead array assay.

BMS-986178 treated CD4 as shown in FIGS. 33B and 33F⁺And CD8⁺T cells produced IL-2 in the presence of Tregs, suggesting that agonism of OX40 via BMS-986178 alleviates Treg suppression. CD4⁺T cells (FIG. 33B) or CD8⁺IL-2 production by T cells (FIG. 33F) in the presence of Tregs all showed a hook effect. On the other hand, at about 1. mu.g/mLBMS-986178, OX40 expression on Tregs from the corresponding cultures (FIGS. 33D and 33H) reached a maximum, whereas>BMS-986178 at 1. mu.g/mL resulted in a decrease in OX40 surface expression.

In addition, IL-2 produced by stimulated CD4+ T cells alone (FIG. 33C) also exhibited a hook effect. In addition, CD4 cultured alone at 0.37. mu.g/mL of BMS-986178⁺T cells (FIG. 33E) and CD8⁺T cells (FIG. 33I) showed the greatest surface expression of OX40>At 1. mu.g/mL of BMS-986178, the expression of OX40 was reduced. On the other hand, stimulated CD8+ T cells showed maximal IL-2 production regardless of the dose of BMS-986178 (fig. 33G).

These data indicate that BMS-986178 alleviates CD4⁺And CD8⁺T cells were suppressed for tregs and had a hook effect relative to dose.

Example 8 in vitro assessment of the relationship between soluble OX40 and receptor occupancy

An ELISA specific for soluble OX40(sOX40) was developed (fig. 16). Briefly, in this assay, a non-blocking OX40 monoclonal antibody (OX40.8) was first immobilized on Meso Scale Discovery (MSD) plates. After blocking the plates with PBS containing 0.5% BSA, culture supernatants were applied to the plates and incubated. After washing, the captured sOX40 was detected using a pre-optimized concentration of biotin-conjugated anti-OX 40 sheep polyclonal antibody (R & D System). After incubation and washing, sulfotag streptavidin was added and the sOX 40-antibody complexes immobilized on the plate were detected by electrochemiluminescence. This assay was used to assess the relationship between sOX40 levels and receptor occupancy. The cells were cultured under the same conditions as described in example 7, except for the following differences: after 4 days of culture, the supernatant was collected and sOX40 quantified using the assay described above.

As shown in fig. 17 and 18, increasing the concentration of OX40.21 induced the expression of OX40 on the surface of CD4+ T cells and sOX40 in the supernatant. At 100% receptor occupancy, the loss of sOX40 was correlated with the loss of cell surface OX40 ("hook" effect). This data suggests sOX40 may serve as a quantitative marker of OX40 agonism and/or T cell activation (readout). Likewise, sOX40 levels were also increased in human patients when treated with OX40.21 monotherapy and OX40.21+ nivolumab (FIG. 19).

anti-CD 28 treated CD4 was determined using a custom developed ELISA as described above⁺Soluble OX40 released by T cells (sOX 40). As shown in figure 35A, this hook effect observed in cells treated with BMS-986178 is unique in that anti-CD 28 induces sOX40 release in a dose-dependent manner after reaching a maximum at higher doses. Further, as shown in FIG. 35B, from CD4⁺Most of the sOX40 released by the T cells bound to BMS-986178. It can be seen that BMS-986178 mediated T cell activation induced sOX40 release, which sOX40 release decreased as OX40RO approached 100%.

Total sOX40 and sOX40 bound by OX40.21 were also determined. Briefly, total T cells were purified from human whole blood using Ficoll gradient centrifugation. CD4+ T cells were isolated from PBMCs using the Miltenyi CD4+ isolation kit. Isolated T cells were cultured in the presence of irradiated CHO-OKT3-CD32A (artificial antigen presenting cells) and tested with serial dilutions of agonist anti-OX 40 antibody, isotype control or anti-CD 28 (clone 28.2). At a preset time point, the supernatant was collected. Soluble OX40 was quantified using the ELISA method described above.

As shown in fig. 20A and 20B, levels of sOX40 correlated with cell surface expression of OX40, cleavage of OX40 was cell activation dependent, but not specific for OX40 agonists, and intact sx 40.21 bound to OX40 correlated with decreased levels of total soluble OX40 (i.e., 100% sOX40 binding when a "hook" effect of sOX40 was detected).

Next, the ability of anti-CD 28 antibodies to rescue the OX 40.21-mediated "hook" effect was tested. Total T cells were purified from human whole blood by Ficoll gradient centrifugation. CD4+ T cells were isolated from PBMCs using the Miltenyi CD4+ isolation kit. Isolated T cells were cultured in the presence of irradiated CHO-OKT3-CD32A, and in the presence of serially diluted agonist anti-OX 40 antibody, serially diluted isotype control alone, or serially diluted isotype control in combination with a constant concentration of anti-CD 28 antibody (clone CD 28.2). In another case, a constant concentration of anti-CD 28 antibody was used alone and serially diluted OX40.21 antibody or isotype control was added after 72 hours (3d) for 18 hours. The supernatant was collected at a predetermined time (D4). Soluble OX40 was quantified using the MSD ELISA described above.

As shown in fig. 21A and 21B, co-stimulation of cells with OX40.21 and anti-CD 28 antibodies had an additive effect on sOX40 levels. However, CD28 co-stimulation did not rescue the OX 40.21-induced "hook" effect on sOX40. Treatment of anti-CD 28 antibody pre-activated cells with OX40.21 was sufficient to induce sOX40 release and a "hook" effect on sOX40 levels was observed. Taken together, these data indicate that sOX40 can be a biomarker of primary activation of human T cells in vitro, as well as in patients treated with immunostimulatory agonists in combination with checkpoint blockers. For patients receiving agonistic antibodies that bind to immunostimulatory receptors as monotherapy or in combination with checkpoint blockers, the measurements of sOX40 can be used as clinical biomarkers to determine their optimal dosage and schedule. Furthermore, the generation of sOX40 is not a mechanism that could explain the loss of OX40 from the cell surface at OX40.21 above 20-40% RO, since sOX40 is also lost at concentrations in the optimal range that results in RO above 20-80%. This suggests that cell surface OX40 and sOX40 are regulated in a similar manner.

Example 9: in vitro assessment of OX40 internalized by OX40

This example evaluates the internalization of OX40 following treatment with blocking (OX40.21) and non-blocking (OX40.8) antibodies. A general schematic of this assay is shown in fig. 22.

More specifically, in vitro generated CD4+ Tregs were activated with CD3/CD28 dynabeads for 48 hours. Serially diluted OX40.21, non-blocking OX40.8, and isotype control were incubated with cells on ice for 2 hours, followed by addition of pH sensitive conjugated anti-human Fc at 37 ℃ for 2 hours. After fixation, cell internalization was analyzed with ArrayScan.

As shown in figure 23, OX40.21 and OX40.8 internalize after cross-linking in Tregs.

In addition, the internalization of OX40 in response to two different doses (0.01nM and 100nM) of BMS-986178 was determined. In this assay, activated Tregs or CD4 will be detected⁺T cells were incubated with isotype control mAb or BMS-986178. Then, pH sensitive dye (pHrodo) conjugated anti-Fc was added. Thereafter, the cells were fixed and read on an ArrayScan VTI (ThermoFisher Scientific).

As shown in figure 36, Tregs or CD4+ T cells treated with 0.01 or 100nM of BMS-986178 internalized drug-bound OX40 in a BMS-986178 concentration-dependent manner.

Example 10: effect of Fc γ R-mediated Cross-linking on the agonistic Activity of OX40.21

This example demonstrates the effect of Fc γ R mediated cross-linking on the agonistic activity of OX 40.21. Briefly, CHO cells were engineered to express the cell membrane bound scFv version of anti-human agonist CD3 clone OKT3, as well as the H131 allele (+ Fc γ R) of human Fc γ RIIa (CD32a-H131), or the absence of the allele (-Fc γ R), designated "OKT 3 scFv" and "hFc γ R", respectively. CHO cells were irradiated to limit their proliferation and cultured with primary human CD 4T cells and varying amounts of OX 40.21. Primary human CD 4T cells were evaluated for T cell proliferation and IFN- γ secretion over a period of 4 days.

As shown in fig. 24, treatment with BMS-986178 induced CD4+ T cell proliferation and IFN- γ secretion in a dose-dependent and cross-link dependent manner. Fc gamma R-mediated cross-linking promotes BMS-986178-mediated IFN-gamma secretion and T cell proliferation. However, when CHO cells lacked CD32a (CD32a-H131), neither proliferation nor IFN- γ was present, suggesting that Fc γ R-mediated cross-linking is required for BMS-986178 activity.

Example 11 characterization of peripheral pharmacodynamic markers induced by anti-OX 40 antibody monotherapy and combination therapy with anti-PD 1

This example demonstrates the induction of certain pharmacodynamic markers in mice treated with OX40.23 monotherapy or with OX40.23 in combination with an anti-PD 1 antibody, as well as in human patients treated with OX40.21 in combination with an anti-PD 1 antibody (nivolumab).

Mice with established CT26 tumors received OX40.23 monotherapy or in combination with anti-PD 1 antibodies. OX40.23 dose escalations started at 0.01mg/kg, and were 3-fold escalated to 90 mg/kg. The dose of anti-PD-1 antibody was 10mg/kg (or a fixed dose of 200 ug/mouse). OX40.23 and anti-PD 1 antibodies were administered on days 6, 13, and 20 on the same schedule. 50ul of whole blood was collected from a single mouse on days 8, 12, 15 and 19. Flow analysis was performed to determine the induction of peripheral pharmacodynamic markers (ICOS, FOXP3, Ki67 and CD44) in CD4+ and CD8+ T cells following treatment with OX40.23-mIgG1 ± anti-PD-1. Fig. 25 is a schematic of a dosing schedule.

As shown in figure 26A, both CD4+ T cells and CD8+ T cells showed dose-dependent up-regulation of activation markers (ICOS, CD44, Ki67) on CD4 effector T cells and CD8+ T cells, while down-regulated at higher doses ("hook" effect).

Likewise, combination therapy increased proliferative (Ki67+) CD8+ T cells (fig. 26B) and decreased FOXP3+ cells in the tumor stroma (fig. 26C) in human patients treated with OX40.21+ anti-PD 1 antibody (nivolumab).

Immunohistochemical analysis of tumor samples from patients resulted in agreement. For example, as shown in fig. 26D, a sample from a 68 year old female endometrial cancer patient who received 3 lines of prior treatment (medroxyprogesterone, letrozole, and carboplatin and paclitaxel) and had partial remission with OX40.21(320mg) + nivolumab (240mg) treatment showed an increase in Ki67+ CD8+ T cell numbers. This combination therapy also reduced FoxP3+ cells in tumor samples from ovarian cancer patients who had reached disease stability. As shown in fig. 26E, a reduction in FoxP3+ cells was observed in one female ovarian serous carcinoma patient (upper panel) that had previously undergone surgery and chemotherapy (carboplatin and paclitaxel) and in one ovarian adenocarcinoma patient (lower panel) that had previously undergone surgery and chemotherapy (carboplatin and paclitaxel).

Example 12: correlation between early T cell activation markers and tumor response to anti-OX 40 and anti-PD 1 combination therapy

This example evaluated the correlation between early T cell activation markers and tumor response to anti-OX 40(OX40.23) and anti-PD 1 combination therapy.

Mice were divided into two groups according to the tumor progression status at day 20. Tumor volume>100mm³The mouse is regarded as a non-responder, and the tumor volume is less than or equal to 100mm³The mouse of (a) is considered a responder.

As shown in fig. 27A and 27B, the percentage of CD44+ CD8+ T cells and Ki67+ CD8+ T cells at day 12 (when tumor volumes were not significantly separated) correlated positively with subsequent tumor response, which may help determine the optimal dose and schedule for combination therapy. Data plotted as absolute change in Ki67+ CD8+ T cells versus maximum% reduction in tumor burden also showed a positive correlation between reduction in tumor burden and proliferating CD8+ T cells (fig. 27C). Likewise, anti-tumor activity was associated with an increase in proliferating Ki67+ CD8+ T cells in human patients treated with OX40.21+ anti-PD 1 antibody (nivolumab) (fig. 27D).

Example 13: effect of increasing doses of anti-ICOS antibodies on tumor growth

This example shows that an agonistic anti-ICOS antibody exhibits reduced efficacy (i.e., a "hook effect") at higher doses in the anti-ICOS + anti-PD 1 combination therapy.

Briefly, mice with established CT26 tumors (average weight about 20mg) were treated with anti-PD-1 monotherapy or in combination with anti-ICOS antibodies. Dose escalation against ICOS started at 0.1mg/kg and was 3-fold escalated to 10mg/kg (or one maximum dose: about 200. mu.g/mouse fixed dose). anti-PD-1 antibody was administered at a dose of 10mg/kg (or one maximum dose: a fixed dose of about 200. mu.g/mouse). The anti-ICOS and anti-PD 1 antibodies were administered on the same schedule following tumor implantation (i.e., every 4 days, starting on day 7).

As shown in figure 28, the anti-ICOS antibody dose at which maximum Tumor Growth Inhibition (TGI) was observed (3mg/kg) was lower than the maximum dose tested (10mg/kg) in the anti-ICOS + anti-PD 1 combination treatment, indicating a decrease in TGI at doses greater than 3 mg/kg. This suggests that agonist antibodies targeting other immunostimulatory receptors, such as ICOS and the like, exhibit a "hook effect" similar to agonist anti-OX 40 antibodies and achieve maximal efficacy at sub-saturating doses.

Example 14: selection of a first human trial dose of OX40.21 using a pharmacokinetic/pharmacodynamic (PK/PD) based approach

The First human trial (FIH) dose of tumor drugs is generally based on the International Conference on harmony guidelines (International Conference on harmony guidelines) which recommends initial doses of FIH as follows: 1/6 for the highest non-severe toxic dose (HNSTD) from non-rodents; alternatively, for biopharmaceuticals with immuno-agonist properties, the recommended dose is the lowest expected biological effect level (MABEL). However, the MABEL-based approach does not provide clinical benefit to the patient. This example describes a PK/PD based approach to select the initial dose of FIH based on anti-tumor efficacy, i.e. the expected pharmacological effect.

Briefly, the half maximal effective concentration of binding (EC) in vitro in human and mouse activated T cells was determined by flow cytometry₅₀) The value is obtained. For mouse PK/PD assays, a mouse surrogate antibody (hamster anti-mouse OX40 agonist monoclonal antibody, reformatted to mouse IgG1(mIgG1) and IgG2a (mIgG2a) isotypes) was used because OX40.21 does not bind to mouse OX40. For anti-tumor efficacy, percent tumor growth inhibition (% TGI) was determined from the median of the areas under the tumor growth curves in the treated and control groups using the mouse MC38 and CT26 colon adenocarcinoma models. PK studies were performed in cynomolgus monkeys using the surrogate antibody and OX 40.21. Prediction of human PK based on monkey data using simple isovelocity scaling (allegory), with clearance and steady state scoresThe power exponent of the cloth measure (Vss) is 0.85 and 1, respectively. Cytokine release was evaluated in vitro using the "dry coat" (drecoat) format (see, e.g., Finco et al. cytokine 2014; 66: 143-55). An adenovirus serotype 5-simian immunodeficiency virus (Ad5-SIV) vaccination study was performed in cynomolgus monkeys with OX40.21, and a1 month repeat administration toxicology study was performed in cynomolgus monkeys with 30 min intravenous (iv) infusions of 30mg/kg, 60mg/kg and 120mg/kg of OX40.21 once a week for 5 weeks.

As shown in Table 6, mouse surrogate antibodies exhibited binding EC similar to OX40.21₅₀The value is obtained.

TABLE 6

Next, PK/PD analysis was performed to determine the maximum drug concentration (C) in the first week_{max (first week)}) Or area under the curve (AUC)_{0-first week}) Associated with anti-tumor efficacy. To predict the efficacy dose in humans, and to be more conservative to broaden patient coverage, this analysis was performed around the antitumor efficacy in the MC38 model, because the model is less sensitive to antitumor efficacy compared to the CT26 model. Since the PK of the mouse surrogate was hamster-derived and significantly affected by immunogenicity in the second week, C in the first week was simulated under various protocols used in the efficacy test based on the assumption that the pharmacological effects of the agonist were driven by Cmax or initial drug exposure_maxAnd AUC data. The exposure-response relationship of mIgG1 and mIgG2 monoclonal antibodies in the mouse MC38 tumor model is shown in FIG. 29.

Corresponding PK experiments were also performed in cynomolgus monkeys, and the results are summarized in table 7.

TABLE 7

Prediction of human T from the above mouse and monkey data_1/2The period is 9 days. By achieving the same AUC in humans as in mice_{0-first week}And C_{max (first week)}The estimated human effective dosage of OX40.21 is 1 mg/kg. The initial dose in humans was selected as 1/4 (i.e., 0.25mg/kg, or 20mg at 80kg body weight) in an estimated effective dose to more efficiently achieve clinically relevant doses.

Additional auxiliary data were obtained to provide information for FIH initial dose selection as follows:

ad5-SIV vaccination studies with OX40.21, showing that at IV doses of 4mg/kg, the enhancement of vaccine-induced T cell responses was found to be minimal when monkeys were administered OX40.21 on days 1 and 28.

Using the "dry coat" format, at the highest concentration tested (10 μ g/well or 33 μ g/mL, approximated using an incubation volume of 0.3mL), OX40.21 did not induce cytokine release or increase activation of expression signatures in human peripheral blood mononuclear cells.

-HNSTD or no adverse effect level of 120 mg/kg/week calculated as one sixth of HNSTD dose of 17mg/kg (based on exposure) or 20mg/kg (based on body weight) by repeated dose toxicology studies in monkeys for 1 month.

Clinical experience with another mouse anti-human OX40 agonist mAb reported in the literature shows that there is no acute toxicity in Cancer patients, even at the highest dose tested (2mg/kg), the drug concentration in humans (≈ 40 μ g/mL) is well maintained throughout the first week of dosing (see Curti et al, Cancer Res 2013; 73: 7189-98). At 2mg/kg, human C_maxAbout 80. mu.g/mL, or reported in vitro binding EC₅₀(48ng/mL or 0.3nM)>1500 times.

Table 8 summarizes C at initial doses of FIH based on PK/PD_maxMargin and additional support data as described above.

TABLE 8

NA: not applicable to

^aDrug concentration in stem-coat cytokine release assaysThe incubation volume (0.3mL) was approximated and the human dose was calculated by multiplying the ineffective drug level by the amount of plasma in 40mL/kg

b binding of EC₅₀Computing margins after normalization of differences in values

In conclusion, the FIH starting dose of the agonist anti-OX 40 antibody OX40.21 was successfully selected and proven to be rational using a PK/PD based approach focusing on pharmacological effects with an anti-tumor efficacy as the goal. The initial dose of FIH chosen (20mg, assuming a body weight of 80 kg) was supported by preclinical in vitro and in vivo toxicology data. This PK/PD based FIH initial dose selection strategy, as well as in vitro and in vivo toxicology outcomes, reflects the intent to minimize the number of cancer patients receiving sub-therapeutic doses while ensuring adequate safety.

Example 15: patient characteristics and treatment-related adverse events in OX40.21 monotherapy and OX40.21+ nivolumab dose escalation trials

This example summarizes baseline demographics, previous treatments, and tumor types and adverse events in patients receiving OX40.21 monotherapy (n ═ 20) Q2W and OX40.21+ nivolumab combination treatment (n ═ 39) Q2W in dose escalation trials. Table 9 summarizes patient characteristics and table 10 summarizes adverse events.

TABLE 9

OX40.21 monotherapy OX40.21+ nivolumab

^aIncluding breast, bladder, cervical, endometrial, gastric, HCC, NSCLC, ovarian, prostate, RCC, and SCCHN CRC ═ colorectal cancer; CTLA-4 ═ cytotoxic T lymphocyte antigen-4; ECOG PS ═ american eastern tumor cooperative group performance status; HCC ═ hepatocellular carcinoma; NSCLC ═ non-small cell lung cancer; procedure for PD-L1Death protein ligand 1; RCC ═ renal cell carcinoma; SCCHN ═ head and neck squamous cell carcinoma

Watch 10

OX40.21 OX40.21+ nivolumab 240mg

Severe adverse events occurred in1 patient each,^a grade 2 pneumonia (OX40.21320mg) and^c grade 3 pneumonia, leading to withdrawal, is considered DLT;^b grade 3 fatigue occurred in1 patient. TRAE: treatment-related adverse events

Maximum tolerated dose was not reached and no treatment-related deaths occurred. The safety of OX40.21+ nivolumab was similar to nivolumab monotherapy.

OX40 co-stimulatory agonists BMS-986178+ -Nivolumab (NIVO) or ipilimumab (IPI) OX40 receptor modulation in a 1/2a phase study in patients with advanced solid tumors

Example 16 comparison of pharmacokinetics of monotherapy and combination therapy

The following example was conducted in accordance with the study design scheme shown in FIG. 39. Administering to a human a monotherapy dose of BMS-986178, an ascending dose of BMS-986178 in combination with 240mg of nivolumab (IV Q2W), or an ascending dose of BMS-986178 in combination with 1mg/kg of ipilimumab (IV Q3W). Patients meeting the criteria in figure 39 were eligible for study.

To evaluate the pharmacokinetics of monotherapy compared to combination therapy, patient blood samples were collected in Cyto-Chex BCT (Streck). After lysis of the erythrocytes, the cells were stained with fluorescently labeled surface marker-specific antibodies. After surface staining, the samples were fixed, permeabilized and then stained with antibodies to intracellular markers. Stained samples were collected on a Beckman Coulter CytoFlex S flow cytometer and the data analyzed using FlowJo software.

In this study, 90 patients received treatment (BMS-986178 monotherapy, n-20; BMS-986178+ NIVO, n-38; BMS-986178+ IPI, n-32). As shown in FIG. 40, PK of BMS-986178 used either alone or in combination with either nivolumab or ipilimumab was linear for BMS-986178 doses of 20 to 320 mg. Thus, the concentration-time characteristics of BMS-986178 can be well described with a linear, two-compartment, zero-order iv model and first-order elimination.

Example 17: whole blood OX40 Receptor Occupancy (RO) assessment of monotherapy and combination (agonist BMS-986178+ -Nivolumab (NIVO) or ipilimumab (IPI)) treatment

To assess whole blood OX40 Receptor Occupancy (RO), patient blood samples were incubated with saturating doses of BMS-986178 to measure total OX40 expression, or without BMS-986178 incubation to measure bound drug, followed by staining and flow cytometry of surface markers C1D1, C1D8, C2D1, or C5D 1.

As shown in FIG. 41, in patients treated with BMS-98617820 mg, the peripheral OX40RO on Tregs was near saturation, reaching saturation at doses ≧ 40 mg.

Next, patients treated with ≥ 40mg BMS-986178 were assessed for the presence or absence of downregulation of peripheral Tregs. To test total OX40 in peripheral Tregs, an assay was developed to determine total sOX40 in patient sera and validated using the MesoScale Discovery (MSD) platform (suitable for the purpose). MSD Gold 96-well streptavidin plates were coated with biotinylated capture antibody and subsequently incubated with patient serum. The captured sOX40 was detected with the ruthenated detection antibody and electrochemiluminescence was measured by msdsecutor meter.

As shown in FIG. 42, down-regulation of Tregs surface OX40 expression was observed in patients treated with ≧ 40mg BMS-986178, at which time RO approached saturation.

It was next determined whether this trend was also observed in individual patients treated with BMS-986178 monotherapy or with BMS-986178 in combination with nivolumab or ipilimumab. As shown in fig. 43A and 43B, marker sOX40 of T cell activation showed time and dose dependent regulation in patients treated with BMS-986178 ± nivolumab or ipilimumab, confirming engagement of BMS-986178 to OX40 target. sOX40 levels were nearly saturated in patients treated with ≧ 160mg of BMS-986178, which is consistent with RO results (FIG. 42) and preclinical observations (data not shown).

Example 18: serum cytokine expression for monotherapy and combination (agonist BMS-986178+ -Nivolumab (NIVO) or ipilimumab (IPI)) treatment

Interferon-gamma (IFN- γ) and IP-10 were determined in patients receiving BMS-986178 monotherapy and combination therapy. Briefly, IFN- γ and IP-10 in patient sera were determined using Luminex-based technology (customized multi-analyte Spectroscopy [ MAP ] panel in combination with various human inflammatory MAP panels [ Myriad RBM ]). As shown in FIGS. 44A and 44B, BMS-986178+ -NIVO or IPI stimulated the production of the TH 1-associated cytokines IFN- γ (FIG. 44A) and the proinflammatory cytokine 10kDaIFN- γ inducible protein IP-10 (FIG. 44B), suggesting peripheral T cell activation. In addition, more of the patients receiving BMS-986178+ NIVO or IPI showed robust increases in IFN-. gamma.and IP-10 production.

BMS-986178+ -NIVO or IPI was also observed to increase the levels of proliferative (Ki67+) CD4+ and CD8+ effector memory T cells. As shown in figures 45A and 45B, patients treated with BMS-986178 ± NIVO or IPI showed increased levels of proliferation (Ki67+) of CD4+ effector memory T cells (figure 45A) and CD8+ PD-1+ terminal effector memory T cells (figure 45B). It can be seen that BMS-986178+ NIVO or IPI showed a more profound increase in proliferative CD4+ and CD8+ effector memory T cells in a greater number of patients than BMS-986178 alone.

Based on the above data, without being limited to a particular mechanism, fig. 37 shows a schematic model of the relationship between BMS-986178 dose, OX40RO, OX40 expression and PD regulation.

Example 19 development and validation of a human Total soluble OX40 biomarker assay

An assay to measure total sOX40 in patient serum was developed and validated using the Meso Scale Discovery (MSD) platform as described in example 17. The assay was validated using biomarker validation methods appropriate for the purpose (fit-for-purpose), including accuracy and precision, dilution linearity/parallelism, specificity (matrix effects and drug interference), stability and selectivity.

The results of the experiment are shown in FIGS. 46A to 46H and tables 11 to 12. As shown in the figure, the measurement range of human serum total sOX40 is 25-20,000 pg/mL. The accuracy of 7 standard curve points (n is 20) is within 98-103%, and CV is less than or equal to 8%. The QC performance is within acceptable limits, and the CVs of LQC, MQC, HQC, and ULOQ are all below 14% (wherein the CV of LLOQ is 26%). Table 11 and table 12. The assay calibrators showed parallelism between BMS-986178 and the commercial calibrators (i.e., OX40-His _ Sino and OX40-Fc _ R & D). Fig. 46A. There was also a good correlation of serum OX40 between two different antibody pairs. Fig. 46B. Dilution linearity/parallelism, specificity, stability and selectivity were all within ± 25% of the expected concentration. Fig. 46C to 46E. Storage and freeze-thaw stability of OX40 are also within performance expectations. And (H). Finally, minimal interference by anti-OX 40 antibody, OX40L fusion protein, nivolumab, and ipilimumab was observed. FIGS. 46F, 46G and 48. These results demonstrate the high accuracy, sensitivity and applicability of the assay to clinical sample analysis.

Table 11: calibration curve verification data summarization

Table 12: QC performance

Once validated, the assay was used to measure serum sOX40 levels in normal healthy volunteers and cancer subjects. As shown in figure 47, sOX40 levels were significantly elevated in cancer patients (head and neck, ovarian, or cervical cancer) compared to normal healthy individuals. This result supports the use of anti-OX 40 antibodies (e.g., BMS-986178) alone or in combination with other therapeutic agents such as nivolumab and ipilimumab to treat cancer.

TABLE 13 summary of sequences

Equivalent scheme:

those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments disclosed herein. Such equivalents are intended to be encompassed by the following claims.

Sequence listing

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245 250 255

Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser

260 265 270

Ala Leu Ala Lys Ile

275

<210>4

<211>183

<212>PRT

<213> Artificial sequence

<220>

<223> human OX40L

<400>4

Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg

1 5 10 15

Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val Ala Ser Val Ile Gln

20 25 30

Gly Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile Cys Leu His Phe Ser

35 40 45

Thr Leu Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val

50 55 60

Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln

65 70 75 80

Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn

85 90 95

Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu

100 105 110

Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln

115 120 125

Leu Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr

130 135 140

Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu

145 150 155 160

Asp Asp Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn

165 170 175

Pro Gly Glu Phe Cys Val Leu

180

<210>5

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223>VHCDR1

<400>5

Ser Tyr Ala Met Tyr

1 5

<210>6

<211>16

<212>PRT

<213> Artificial sequence

<220>

<223>VHCDR2

<400>6

Ala Ile Asp Thr Asp Ala Gly Thr Phe Tyr Ala Asp Ser Val Arg Gly

1 5 10 15

<210>7

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>VHCDR3

<400>7

Leu Gly Glu Gly Tyr Phe Phe Asp Tyr

1 5

<210>8

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>VLCDR1

<400>8

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210>9

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>VLCDR2

<400>9

Asp Ala Ser Asn Arg Ala Thr

1 5

<210>10

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>VLCDR3

<400>10

Gln Gln Arg Ser Asn Trp Pro Pro Thr

1 5

<210>11

<211>117

<212>PRT

<213> Artificial sequence

<220>

<223>VH

<400>11

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Asp Thr Asp Ala Gly Thr Phe Tyr Ala Asp Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Leu Gly Glu Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210>12

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223>VL

<400>12

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>13

<211>446

<212>PRT

<213> Artificial sequence

<220>

<223>HC

<400>13

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Asp Thr Asp Ala Gly Thr Phe Tyr Ala Asp Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala

85 90 95

Arg Leu Gly Glu Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210>14

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223>LC

<400>14

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>15

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic

<400>15

Asp Val Val Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp Cys Asn Leu

1 5 10 15

Arg

<210>16

<211>440

<212>PRT

<213> Artificial sequence

<220>

<223> heavy chain nivolumab

<400>16

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 4045

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>17

<211>214

<212>PRT

<213> Artificial sequence

<220>

<223> light chain nivolumab

<400>17

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr AspAla Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly GluCys

210

<210>18

<211>113

<212>PRT

<213> Artificial sequence

<220>

<223> variable heavy chain region nivolumab

<400>18

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210>19

<211>107

<212>PRT

<213> Artificial sequence

<220>

<223> light chain variable region nivolumab

<400>19

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>20

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223>HCDR1 nivolumab

<400>20

Asn Ser Gly Met His

1 5

<210>21

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>HCDR2 nivolumab

<400>21

Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>22

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223>HCDR3 nivolumab

<400>22

Asn Asp Asp Tyr

1

<210>23

<211>11

<212>PRT

<213> Artificial sequence

<220>

<223>LCDR1 nivolumab

<400>23

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210>24

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>LCDR2 nivolumab

<400>24

Asp Ala Ser Asn Arg Ala Thr

1 5

<210>25

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>LCDR3 nivolumab

<400>25

Gln Gln Ser Ser Asn Trp Pro Arg Thr

1 5

<210>26

<211>118

<212>PRT

<213> Artificial sequence

<220>

<223> heavy chain variable region ipilimumab

<400>26

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210>27

<211>108

<212>PRT

<213> Artificial sequence

<220>

<223> light chain variable region

<400>27

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>28

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223>HCDR1 ipilimumab

<400>28

Ser Tyr Thr Met His

1 5

<210>29

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223>HCDR2 ipilimumab

<400>29

Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>30

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>HCDR3 ipilimumab

<400>30

Thr Gly Trp Leu Gly Pro Phe Asp Tyr

1 5

<210>31

<211>12

<212>PRT

<213> Artificial sequence

<220>

<223>LCDR1 ipilimumab

<400>31

Arg Ala Ser Gln Ser Val Gly Ser Ser Tyr Leu Ala

1 5 10

<210>32

<211>7

<212>PRT

<213> Artificial sequence

<220>

<223>LCDR2 ipilimumab

<400>32

Gly Ala Phe Ser Arg Ala Thr

1 5

<210>33

<211>9

<212>PRT

<213> Artificial sequence

<220>

<223>LCDR3 ipilimumab

<400>33

Gln Gln Tyr Gly Ser Ser Pro Trp Thr

1 5

Claims (75)

1. A method of treating cancer, comprising administering to a subject in need thereof an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the agonistic antibody is administered in an amount or frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

2. A method of reducing or depleting the number of T regulatory cells in a tumor in a subject having cancer, comprising administering to the subject an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the agonistic antibody is administered in an amount or frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

3. A method of increasing IL-2 and/or IFN- γ production in T cells in a patient having cancer, comprising administering to the subject an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the agonistic antibody is administered in an amount or frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

4. A method of stimulating an immune response in a subject having cancer comprising administering to the subject an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the agonistic antibody is administered in an amount or frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

5. A method of inhibiting tumor cell growth in a subject having cancer, the method comprising administering to the subject an agonistic antibody that specifically binds to an immunostimulatory receptor, wherein the agonistic antibody is administered in an amount or frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

6. The method of any one of the preceding claims, wherein the agonistic antibody is administered in an amount or frequency sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 80%.

7. The method of claim 5, wherein said agonistic antibody is administered in an amount sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 70%.

8. The method of claim 5, wherein the agonistic antibody is administered in an amount sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 60%.

9. The method of claim 5, wherein the agonistic antibody is administered in an amount sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 50%.

10. The method of claim 5, wherein the agonistic antibody is administered in an amount sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 40%.

11. The method of any one of claims 1-10, wherein receptor occupancy and/or receptor occupancy range is measured on day 1 after cycle 1 of a treatment regimen with the agonistic antibody.

12. The method of any one of the preceding claims, wherein the immunostimulatory receptor is selected from the group consisting of: LFA-1(CD11a/CD18), CD2, CD7, CD30, CD40, CD54, CD160, BAFFR, HVEM, LIGHT, NKG2C, SLAMF7 and NKp 80.

13. The method of any one of claims 1-12, wherein the immunostimulatory receptor is a co-stimulatory receptor.

14. The method of claim 13, wherein the co-stimulatory receptor is a member of the tumor necrosis factor receptor superfamily.

15. The method of any one of the preceding claims, wherein the agonistic antibody is selected from IgG1, IgG2, IgG3, IgG4, or a variant thereof.

16. The method of claim 15, wherein the agonistic antibody is an IgG1 antibody.

17. The method of claim 16, wherein the agonistic antibody comprises an Fc with enhanced binding to an activated fcyr.

18. The method of any one of the preceding claims, wherein the agonistic antibody is a human antibody, a humanized antibody, or a chimeric antibody.

19. The method of any one of the preceding claims, wherein the agonistic antibody is a bispecific antibody.

20. The method of any one of the preceding claims, wherein the cancer is selected from the group consisting of: bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system tumor, lymphoma, leukemia, myeloma, sarcoma, non-small cell lung cancer, and virus-associated cancer.

21. The method of any one of the preceding claims, wherein the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer.

22. The method of any one of the preceding claims, further comprising administering one or more additional therapies.

23. The method of claim 22, wherein the one or more additional therapies comprise an antibody or a small molecule.

24. The method of claim 23, wherein the one or more additional therapies comprise an anti-PD 1 antibody, LAG-3 antibody, CTLA-4 antibody, PD-L1 antibody, or anti-TGF antibody.

25. The method of any one of the preceding claims, wherein the agonistic antibody is administered prior to administration of the one or more additional therapies.

26. The method of any one of the preceding claims, wherein the agonistic antibody is administered after administration of the one or more additional therapies.

27. The method of any one of the preceding claims, wherein the agonistic antibody is administered concurrently with the one or more additional therapies.

28. The method of any one of the preceding claims, wherein the agonistic antibody is formulated as a pharmaceutical composition.

29. A method of selecting an effective dose of a therapeutic agonistic antibody that specifically binds an immunostimulatory receptor or a schedule of antibody administration for treating a subject having cancer, the method comprising:

(a) administering the agonistic antibody to an animal model;

(b) obtaining a sample from the animal model;

(c) determining a receptor occupancy or receptor occupancy range for the agonistic antibody in the sample;

(d) using the receptor occupancy or receptor occupancy range obtained from step (c) to predict or predict an expected receptor occupancy or receptor occupancy range in the subject; and

(e) selecting a dose or antibody administration schedule sufficient to achieve and/or maintain a receptor occupancy of less than about 80% in the subject based on the expected receptor occupancy obtained in step (d).

30. The method of claim 29, wherein the amount of agonistic antibody is selected sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 80%.

31. The method of claim 29, wherein the amount of agonistic antibody is selected sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 70%.

32. The method of claim 29, wherein the amount of agonistic antibody is selected sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 60%.

33. The method of claim 29, wherein the amount of agonistic antibody is selected sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 50%.

34. The method of claim 29, wherein the amount of agonistic antibody is selected sufficient to achieve and/or maintain a receptor occupancy range of about 20% to about 40%.

35. A method of treating cancer comprising administering an effective amount of a therapeutic agonistic antibody that specifically binds to an immunostimulatory receptor, or a pharmaceutical composition comprising the same, wherein the amount of agonistic antibody to be administered has been selected according to the method of any one of claims 29-34.

36. A method of monitoring the level of therapeutic agonistic antibodies that specifically bind to an immunostimulatory receptor in a subject undergoing treatment for cancer, comprising

(a) Obtaining a sample from a subject;

(b) determining the receptor occupancy of said agonistic antibody in the sample;

(c) decreasing the amount or frequency of administration of the agonistic antibody to the subject if the receptor occupancy is greater than about 80%, or increasing the amount or frequency of the agonistic antibody if the receptor occupancy is less than about 20%;

(d) optionally repeating steps (a) - (c) until an occupancy of about 20% to about 80% of the receptors is reached and/or maintained.

37. The method of claim 36, wherein in step (c), the amount or frequency of agonistic antibodies is reduced if receptor occupancy is greater than about 70%.

38. The method of claim 36, wherein in step (c), the amount or frequency of agonistic antibodies is reduced if receptor occupancy is greater than about 60%.

39. The method of claim 36, wherein in step (c), the amount or frequency of agonistic antibodies is reduced if receptor occupancy is greater than about 50%.

40. The method of claim 36, wherein in step (c), the amount or frequency of agonistic antibodies is reduced if receptor occupancy is greater than about 40%.

41. The method of claim 36, wherein in step (c), the amount or frequency of agonistic antibodies is increased if the receptor occupancy is less than about 30%.

42. The method of claim 36, wherein in step (c), the amount or frequency of agonistic antibodies is increased if receptor occupancy is less than about 40%.

43. The method of claim 36, wherein in step (c), the amount or frequency of agonistic antibodies is increased if receptor occupancy is less than about 50%.

44. The method of claim 36, wherein in step (c), the amount or frequency of agonistic antibodies is increased if receptor occupancy is less than about 60%.

45. The method of claim 36, wherein in step (d), the amount or frequency of agonistic antibodies is adjusted until about 20% to about 70% receptor occupancy is achieved and/or maintained.

46. The method of claim 36, wherein in step (d), the amount or frequency of agonistic antibodies is adjusted until a receptor occupancy of about 20% to about 60% is achieved and/or maintained.

47. The method of claim 36, wherein in step (d), the amount or frequency of agonistic antibodies is adjusted until about 20% to about 50% receptor occupancy is achieved and/or maintained.

48. The method of claim 36, wherein in step (d), the amount or frequency of agonistic antibodies is adjusted until about 20% to about 40% receptor occupancy is achieved and/or maintained.

49. A method of treating cancer comprising administering to a subject in need thereof an agonistic antibody that specifically binds to an immunostimulatory receptor and an additional therapy, wherein the additional therapy is administered at a fixed frequency, the agonistic antibody being administered at a dose and frequency sufficient to achieve and/or maintain a receptor occupancy of less than about 80%.

50. The method of claim 49, wherein the dose or frequency of the agonistic antibody is determined using the method of claim 29.

51. The method of any one of claims 29-50, wherein the immunostimulatory receptor is a co-stimulatory receptor.

52. The method of claim 51, wherein the co-stimulatory receptor is a member of the tumor necrosis factor receptor superfamily.

53. A method of determining the effectiveness of a cancer treatment in a subject administered a therapeutic agonist antibody that specifically binds to an immunostimulatory receptor, the method comprising measuring the level of soluble OX40 in the subject.

54. The method of claim 53, wherein the level of soluble OX40 is determined by a total soluble OX40 biomarker assay, such as on a Meso Scale Discovery platform.

55. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of each of:

(a) an anti-OX 40 antibody comprising the CDR1, CDR2, and CDR3 domains of the heavy chain variable region having the sequence shown in SEQ ID NO. 11, and the CDR1, CDR2, and CDR3 domains of the light chain variable region having the sequence shown in SEQ ID NO. 12, and

(b) an anti-PD-1 antibody which comprises the CDR1, CDR2 and CDR3 domains of the heavy chain variable region having the sequence shown in SEQ ID NO. 18 and the CDR1, CDR2 and CDR3 domains of the light chain variable region having the sequence shown in SEQ ID NO. 19, and

wherein the method comprises at least one administration cycle, wherein the cycle is a twelve week period, wherein, for each cycle in the at least one cycle, one dose of the anti-OX 40 antibody is administered at a dose of 20, 40, or 80mg and three doses of the anti-PD-1 antibody are administered at a dose of 480 mg.

56. The method of claim 55, wherein the anti-OX 40 antibody and anti-PD-1 antibody are administered at the following doses:

(a)20mg of anti-OX 40 antibody and 480mg of anti-PD-1 antibody;

(b)40mg of anti-OX 40 antibody and 480mg of anti-PD-1 antibody; or

(c)80mg of anti-OX 40 antibody and 480mg of anti-PD-1 antibody.

57. The method of claim 55 or 56, wherein the anti-PD-1 antibody and anti-OX 40 antibody are formulated for intravenous administration.

58. The method of any one of claims 55-57, wherein the anti-PD-1 and anti-OX 40 antibodies are formulated together.

59. The method of any one of claims 55-57, wherein the anti-PD-1 antibody and anti-OX 40 antibody are formulated separately.

60. The method according to any one of claims 55 to 59, wherein the anti-OX 40 antibody is administered prior to administration of the anti-PD-1 antibody.

61. The method according to claim 60, wherein the anti-OX 40 antibody is administered within about 30 minutes before the anti-PD-1 antibody is administered.

62. The method according to any one of claims 55 to 59, wherein the anti-OX 40 antibody is administered after administration of the anti-PD-1 antibody.

63. The method according to any one of claims 55 to 59, wherein the anti-OX 40 antibody is administered simultaneously with the anti-PD-1 antibody.

64. The method of any one of claims 55 to 63, wherein the treatment consists of up to 9 cycles.

65. A method according to any one of claims 55 to 64, wherein the anti-OX 40 antibody is administered on day 1 of each cycle.

66. The method according to any one of claims 55 to 65, wherein the anti-PD-1 antibody is administered on days 1, 29 and 57 of each cycle.

67. A method according to any one of claims 55 to 66, wherein said treatment produces at least one therapeutic effect selected from: tumor size reduction, number of metastases reduction over time, complete remission, partial remission and disease stabilization.

68. The method according to any one of claims 55 to 67, wherein the cancer is selected from bladder cancer, cervical cancer, renal cell carcinoma, testicular cancer, colorectal cancer, lung cancer, head and neck cancer, and ovarian cancer.

69. The method of claim 68, wherein the cancer is bladder cancer.

70. A method according to any one of claims 55 to 69, wherein the anti-OX 40 antibody comprises

(a) A heavy chain variable region CDR1 comprising the sequence set forth in SEQ ID NO. 5;

(b) a heavy chain variable region CDR2 comprising the sequence set forth in SEQ ID NO. 6;

(c) a heavy chain variable region CDR3 comprising the sequence set forth in SEQ ID NO. 7;

(d) a light chain variable region CDR1 comprising the sequence set forth in SEQ ID NO. 8;

(e) a light chain variable region CDR2 comprising the sequence set forth in SEQ ID NO. 9; and

(f) light chain variable region CDR3 comprising the sequence shown in SEQ ID NO. 10.

71. The method of claim 70, wherein the anti-OX 40 antibody comprises: a heavy chain variable region comprising the sequence shown in SEQ ID NO. 11 and a light chain variable region comprising the sequence shown in SEQ ID NO. 12.

72. The method of claim 71, wherein the anti-OX 40 antibody comprises: a heavy chain comprising the sequence shown in SEQ ID NO. 13, and a light chain comprising the sequence shown in SEQ ID NO. 14.

73. The method of any one of claims 55 to 72, wherein the anti-PD-1 antibody comprises:

(a) a heavy chain variable region CDR1 comprising the sequence set forth in SEQ ID NO. 20;

(b) heavy chain variable region CDR2 comprising the sequence shown in SEQ ID NO 21.

(c) Heavy chain variable region CDR3 comprising the sequence shown in SEQ ID NO. 22.

(d) A light chain variable region CDR1 comprising the sequence set forth in SEQ ID NO. 23;

(e) a light chain variable region CDR2 comprising the sequence set forth in SEQ ID NO. 24; and

(f) light chain variable region CDR3 comprising the sequence set forth in SEQ ID NO. 25.

74. The method of claim 73, wherein the anti-PD-1 antibody comprises: a heavy chain variable region comprising the sequence shown in SEQ ID NO 18 and a light chain variable region comprising the sequence shown in SEQ ID NO 19.

75. The method of claim 74, wherein the anti-PD-1 antibody comprises: a heavy chain comprising the sequence shown in SEQ ID NO 16, and a light chain comprising the sequence shown in SEQ ID NO 17.

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